Virus-like particles for the induction of autoantibodies

ABSTRACT

The invention described herein relates to compositions and methods for stimulating immune responses in vivo against a tolerogen. Novel biotechnological tools, pharmaceuticals, therapeutics and prophylactics, which concern chimeric or conjugated virus-like particles, and methods of use of the foregoing are provided for the study of B cell tolerance and the treatment or prevention of human diseases, which involve the onset of B cell tolerance, such as chronic viral infection, chronic inflammatory disease, and neoplasia.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation and claims the benefit ofpriority of U.S. patent application No. 09/835,124 filed Apr. 13, 2001,which is a continuation and claims the benefit of priority ofInternational Application No. PCT/US99/24548 having international filingdate of Oct. 20, 1999 designating the United States of America andpublished in English, which claims the benefit of priority of U.S.Provisional Patent Appl. No. 60/105,132 filed Oct. 21, 1998, all ofwhich are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods forstimulating a B cell immune response in vivo. Novel biological tools,therapeutics, and prophylactics comprising chimeric or conjugatedvirus-like particles and methods of use of the foregoing are providedfor the study, treatment, and prevention of human disease.

BACKGROUND OF THE INVENTION

[0003] It is well established that host immune defenses come into playat various stages of human disease. During viral infection, for example,antibodies stimulated in response to previous immunization . mayneutralize incoming viruses prior to attachment and penetration ofsusceptible target cells. In the event that cells become infected anddisplay virus-associated antigens on their surfaces, cellular immuneresponses may also be activated. In this latter case, cytotoxic T cellscan kill infected cells, thereby limiting progression of the infection.These humoral and cellular immune responses are commonly mounted againstinfection by a wide variety of viruses, including viruses having DNA orRNA genomes and outer coats composed of protein capsids or membraneenvelopes.

[0004] The fact that animals can mount vigorous immune responses to mostforeign antigens without similarly responding to components of their owntissues suggested to Burnet and Fenner (The Production of Antibodies,Macmillan Co., Melbourne (1949)) that the immune system must haveevolved some mechanism for distinguishing self from non-self. A state ofself-tolerance undoubtedly exists for central antigens to which theimmune system is normally exposed. (See Siskind, G., FundamentalImmunology ed. W. E. Paul, Raven Press, New York, Ch. 20 (1984)). A“central antigen” is a self antigen that ordinarily is exposed to cellsof the immune system, whereas a “peripheral antigen” is a self antigenthat ordinarily is shielded from contact with cells of the immunesystem, for example by physical separation. Failure of the immune systemto mount responses against certain components of the eye, brain andtestes, for example, results from segregation of these tissues from thehost immune system rather than from self-tolerance. Indeed, autoimmuneresponses can occur when the physical “barriers” that maintain theseperipheral tissue antigens separate from immune surveillance arecompromised. Remarkably, the vertebrate genome possesses all of theinformation needed to produce antibodies directed against a selfantigen; and spontaneously generated antibodies to many self antigenscan routinely be detected. However, these antibodies are low titer, lowavidity and of the IgM class.

[0005] Several investigators believe that self-tolerance involves theimmune system “learning” to distinguish self and non-self components, anevent that occurs before maturing at around the time of birth. It hasbeen speculated that exposure of the lymphoid system to self antigensduring fetal development, for example, is a critical phase fordeveloping tolerance to self antigens. According to other models,lymphocytes expressing cell surface receptors specific for the selfantigen are eliminated, rendered incapable of activation, or are“tolerized” to the antigen.

[0006] The term “B cell tolerance” is often used to describe a state inwhich the immune system ineffectively responds to the presence of anantigen (e.g., a self antigen) or, more particularly, when the B cellsof the immune system fail to mount a response to an antigen.Accordingly, an antigen that is normally exposed to B cells yet fails toinduce a high titer antibody response or that is associated with anormal non-response by B cells (e.g., a self antigen) is referred to asa “tolerogen” because the immune system “tolerates” its presence.Clearly, self antigens are tolerogens but foreign antigens can alsobecome tolerogens when B cells fail to sufficiently respond to theantigen. Some investigators believe, for example, that chronic viralinfections occur (e.g., viral persistence in infants born to Hepatitis Bvirus (HBV) carrier mothers) because the immune system has becometolerized to viral antigens. (Takashima et al., Immunology, 75:398(1992)). Tolerogens are not necessarily entire molecules but can beportions of molecules (e.g., peptide fragments of proteins), inpotentially immunodominant regions of a molecule. Although investigatorshave had success in inducing tolerance in animals by various techniques,our understanding of ways to generate antibodies to tolerogens is in itsinfancy.

SUMMARY OF THE INVENTION

[0007] The inventors have discovered compositions and methods ofincreasing the titers of antibodies to tolerogens (e.g., self antigensand foreign antigens) over those titers routinely generatedspontaneously or after conventional methods of vaccination. In severalembodiments, the break in B cell tolerance is accomplished by using asupport or capsomeric structure having an ordered assembly of subunitsor capsid proteins joined to at least one B cell epitope of a tolerogen,wherein the tolerogen is presented in a regular, repetitive array. Insome aspects of the invention, the tolerogen and the viral capsidprotein are derived from different organisms, viruses, or infectiousagents. The support can be a bead, a lipid membrane, or a proteinpolymer. The capsomeric structure can have icosohedral or helicalsymmetry. In desirable compositions, however, the capsomeric structureis comprised of viral capsid proteins that self-assemble to form anorganized structure referred to as “virus-like particle,” or VLPs.

[0008] In some embodiments, the viral capsid proteins are hybridmolecules or are otherwise modified. Thus, some embodiments are“chimeric virus-like particles (VLPs)” and others are “conjugatedvirus-like particles (VLPs)”, wherein “chimeric VLPs” have a tolerogenjoined to the viral capsid protein (or its homolog) by geneticengineering (e.g., creation of a tolerogen/capsid protein fusion) and“conjugated VLPs” have a tolerogen joined to the viral capsid protein(or its homolog) by way of chemical, physical or other modification ofthe capsid protein or tolerogen or both (e.g., biotin/streptavidin,biotin/avidin, other ligand/receptor sequences). Thus, aspects of theinvention include a composition comprising a support having an orderedassembly of subunits and at least one B cell epitope of a tolerogenjoined to the support so as to form a tolerogen-presenting immunogen,wherein the tolerogen-presenting immunogen displays the tolerogen in aregular, repetitive array. Other compositions of the invention comprisea capsomeric structure having a symmetrical assembly of capsid proteinsand at least one B cell epitope of a tolerogen joined to the capsomericstructure so as to form a tolerogen presenting virus-like particle(VLP), wherein the tolerogen presenting VLP displays the tolerogen in anordered, repetitive array. Another embodiment of the invention concernsan isolated complex comprising one of these compositions joined to acell of the immune system. Further, pharmaceuticals comprising thesecompositions are embodiments of the invention.

[0009] Methods of generating antibodies to a tolerogen are also part ofthe invention. By one method, antibodies to a tolerogen are generated byidentifying a subject in need of antibodies to a tolerogen and providingto the subject a sufficient amount of one of the compositions describedabove so as to generate antibodies to the tolerogen. Another approachinvolves the identification of agents that generate auto-antibodies.Accordingly, one of the compositions above is provided to a subject,antibodies are isolated from the subject, the titer of the antibodiesisolated in step (b) that bind to the tolerogen are determined and theagent is identified by the ability to generate high titer antibodies.Additionally, a method of generating monoclonal antibodies to atolerogen is within the scope of the invention. By this approach, one ofthe compositions described above is provided to a subject and ahybridoma is made with a B cell from the subject. Other methods includea method of enhancing the production of antibodies to a normallyimmunogenic compound comprising the steps of selecting an antigen thatgenerates a low titer antibody response in a subject, joining thisantigen to a modified VLP so as to form a conjugated VLP, wherein theconjugated VLP displays the antigen in a regular repetitive array, andproviding the conjugated VLP to a subject and thereby generating hightiter antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIGS. 1A and 1B are line graphs showing serum antibody reactivityin an ELISA assay. FIG. 1A shows IgG antibody reactivity to BSA-coupledCCR5 peptide. FIG. 1B shows IgG antibody reactivity to BPV-1 VLPs.Symbols represent results using sera from mice inoculated with L1-CCR5particles ( ), denatured L1-CCR5 particles ( ), or BPV-1 VLPs (

) in the presence of Freund's adjuvant, or L1-CCR5 particles in theabsence of adjuvant ( ).

[0011] FIGS. 2A-2G are histograms illustrating flow cytometric analysisof antibody binding to transiently transfected HeLa-MAGI cells.Constructs encoding CCR5 DNA (thick solid line) or, as a control forbackground staining, vector alone (shaded histogram), were transfectedinto the cells 2 days prior to staining. (2A -2D). Cells transfectedwith mouse CCR5 or vector DNA. (2E-2G). Cells transfected with ahuman/mouse CCR5 chimera (HMHH) or vector DNA. Cells were incubated withpurified IgG from L1-CCR5 immunized mice (2A and 2E), purified IgG fromBPV-1 VLP immunized mice (2B and 2F), or purified IgG from KLH coupledCCR5 peptide immunized mice (2D). As a control, cells were also stainedwith a flourescein-labelled monoclonal antibody against the 2nd EC loopof human CCR5 (2C and 2F).

[0012]FIG. 3 is a bar graph representing displacement of iodinated humanRANTES by sera. HeLa-MAGI cells were transiently transfected with mCCR5.Three days after transfection, cells were incubated with 0.5 nMiodinated RANTES in the absence or presence of dilutions of mouse sera.Maximally bound iodinated RANTES was determined by assaying for bindingin the absence of sera, and corresponds to approximately 2550 cpm(indicated by the dashed line). Non-specific binding of iodinated RANTES(approximately 1300 cpm) was determined by assaying for binding in a1000-fold excess (500 nM) of cold (non-iodinated) human RANTES. Datarepresents the average of duplicate wells from one experiment. Thisassay was repeated on two occasions to ensure reproducibility.

[0013]FIG. 4 is a line graph showing inhibition of HIV-1 BaL infectionusing dilutions of L1-CCR5 sera, BPV-1 VLP sera, or a monoclonalantibody against the second EC loop of human CCR5 (mAB182). Sera waspooled from three animals. HeLa-MAGI cells, an HIV-1 indicator cell linein which the nuclei of infected cells stain blue, were transientlytransfected with a human-mouse CCR5 chimera (HMHH), which contains thefirst EC loop of mouse CCR5 in a background of the human CCR5 gene.Three days after transfection, cells were incubated with dilutions ofpooled mouse sera or antibody for 30 minutes at 4°0 C. Cells were thenchallenged with the M-tropic isolate HIV-1 BaL. Three days afterinfection, infected cells were scored by counting the number of bluecells in each well. Inhibition of HIV-1 BaL infection was determined bycomparing the number of blue (infected) nuclei in the presence of seraversus the number of blue nuclei in the absence of sera. Data representsthe average of duplicate wells from one experiment. To ensurereproducibility, this assay was repeated on at least two otheroccasions, with similar results. Sera from (ν) L1-CCR5 inoculated mice,(μ) BPV-1 VLP inoculated mice, or (A) mAB182.

[0014]FIG. 5 is a line graph that shows primate serum antibodyreactivity in an ELISA assay. Symbols represent results using sera frommacaques inoculated with L1-CCR5 particles with adjuvant ( ) orwild-type BPV-1 VLPs in the absence of adjuvant ( ).

[0015]FIG. 6 is a line graph that shows the binding of streptavidin tobiotinylated and non-biotinylated VLPs. Symbols represent (˜)biotinylated VLPs conjugated with wild type Streptavidin (SA), ( )biotinylated VLPs conjugated with SA-TNF-α, (ν) Non-biotinylated VLPsconjugated with wild type SA, and () non-biotinylated VLPs conjugatedwith SA-TNF-α.

[0016]FIG. 7 is a bar graph that shows the results of a TNF-αcytotoxicity assay. Sera from mice inoculated with a streptavidin-TNF-αfusion protein (SA-TNF-α) joined to a biotinylated VLP was incubatedwith a TNF-α sensitive cell line (L929) in the presence of TNF-α. Serafrom mice innoculated with streptavidin joined to biotinylated VLP wasused as a control. The ability of the cells. Serum from SA-TNF-αinoculated mice (e.g., at a 5% concentration) demonstrated a three-foldincrease in the number of surviving cells when compared to backgroundlevels.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The invention described herein concerns compositions and methodsof increasing the titers of antibodies to “tolerogens,” including selfantigens and foreign antigens, over those titers routinely generatedspontaneously or after conventional methods of vaccination. By “lowtiter antibody response” is meant a B cell response that results in aninsufficient amount of antibodies to mount a physiologically effectivein vivo immune response, whereas, a “high titer antibody response”refers to a sufficient amount of antibodies to mount a physiologicallyeffective immune response in vivo The terms “low titer antibodyresponse” and “high titer antibody response” are also defined accordingto the concentration and avidity of the antibody produced. That is,whether an antigen produces a “low titer antibody response” or a “hightiter antibody response” depends on the dilution of antibody containingsera at which antigen is no longer detectable in an ELISA assay, wherein200 ng of target antigen is typically used with a 1:1000 dilution ofsecondary antibody. Thus, a “low titer antibody response” is typicallyless than about a 1:10000 dilution under the conditions for ELISAdescribed above and a “high titer antibody response” is typicallygreater than or equal to a 1:10000 dilution. It should be understoodthat the term “tolerogen” is used throughout this disclosure to refer toa self antigen or foreign antigen (peptide, nucleic acid, carbohydrate,or lipid) that is either associated with complete B cellnon-responsiveness or limited B cell responsiveness in that the antigenelicits only a low titer antibody response that does not substantiallyaffect the normal in vivo activity of the antigen.

[0018] In several embodiments, the break in B cell tolerance isaccomplished by using a support or capsomeric structure having anordered assembly of subunits or capsid proteins joined to at least one Bcell epitope of a tolerogen, wherein the tolerogen is presented in aregular, repetitive array. In some aspects of the invention, thetolerogen and the viral capsid protein are derived from differentorganisms, viruses, or infectious agents. The support can be a bead, alipid membrane, or a protein polymer. The capsomeric structure can haveicosohedral or helical symmetry. In desirable compositions, however, thecapsomeric structure is comprised of viral capsid proteins thatself-assemble to form an organized structure. Such viral capsidassemblies are referred to as “virus-like particle,” or VLPs.

[0019] In some embodiments, the viral capsid proteins are hybridmolecules or are otherwise modified. The term “virus-like particle” or“capsomeric structure” is often used to refer to an organized structurecomprising self-assembling ordered arrays of capsid proteins that do notinclude a viral genome. In this respect, some embodiments are “chimericvirus-like particles (VLPs)” and others are “conjugated virus-likeparticles (VLPs)”. The term “chimeric VLP” refers to a VLP where thetolerogen is joined to the viral capsid protein (or its homolog) bygenetic engineering (e.g., creation of a tolerogen/capsid proteinfusion). Thus, the tolerogen/capsid protein fusion is often referred toas a “hybrid coat protein” because the viral coat protein is chimerizedwith an amino acid sequence from the B cell epitope of a tolerogen.According to the nomenclature used herein, a hybrid coat protein isidentified by the name of the viral coat protein and the source of thetolerogen that is displayed in connection with the viral coat protein.The term “conjugated VLP” is used to refer to a VLP where the tolerogenis joined to the viral capsid protein (or its homolog) by way ofchemical, physical or other modification of the capsid protein ortolerogen or both (e.g., biotin/streptavidin, biotin/avidin, otherligand/receptor sequences).

[0020] The hybrid coat protein can incorporate the amino acid sequenceof the tolerogen within its primary structure, as by inserting the aminoacid sequence of the tolerogen into the amino acid sequence of the viralcoat protein, or by replacing the amino acid sequence of the viral coatprotein with the amino acid sequence of the tolerogen. The site ofchimerization oftentimes depends on the outer surface of the VLP andregions of the viral coat protein that are involved in self-assembly.This site can correspond to the site of a virus neutralizing epitope,for example. It is to be understood that the hybrid coat protein cantake the form of a single coat protein in certain embodiments of theinvention, a capsomere (5 coat proteins arranged in a pentamer) in otherembodiments, or a VLP composed of multiple capsid proteins arranged as aparticulate structure in preferred embodiments.

[0021] The viral capsid protein that comprises the capsomeric structureof a VLP can be from many different types of viruses but desirableembodiments have proteins that are found in a virus having anicosohedral structure (e.g., T=7) and viruses whose natural reservoirhost is mammal and viruses selected from the families papillomavirinae,polyomavirinae, or parvoviridae. Preferred compositions have a capsidassembly comprising a plurality of papillomavirus hybrid or modified L1proteins.

[0022] By employing the chimeric and conjugated VLP technology disclosedherein, several approaches can be used to join a tolerogen to a supportso as to create many novel compositions. In most embodiments, thecomposition is a “multimeric” support in that more than one tolerogenmolecule is attached to the support. In some embodiments, however, a“multimerized” support is provided in that the tolerogen portion of thecomposition comprises a plurality of the same tolerogen domain fused intandem. Further, multimeric compositions having multimerized tolerogensare also embodiments of the invention. In other embodiments, thecomposition is a “composite” support in that more than one type oftolerogen is presented. One of skill in the art will also appreciatethat composite supports can be multimeric and can include multimerizedtolerogens. Preferably, the multimeric compositions, multimerizedcompositions, and composite compositions and combinations thereof jointolerogens to the support in a manner that optimizes presentation tocells of the immune system. For example, the embodiments present thetolerogens in an ordered, closely spaced, repetitive array.Additionally, some embodiments include linkers engineered between thesupport and the viral capsid protein (or its homolog) or between thetolerogen and the viral capsid protein (or its homolog) or both so as toreduce steric hindrance and encourage optimal immune response. Thus,some compositions have a viral capsid protein (or its homolog) or atolerogen or both that are joined to the support by way of a linker.

[0023] Many different tolerogens can be joined to the support includingpeptides, nucleic acids, carbohydrates, and lipids. In some embodiments,the tolerogen is a self antigen. For example, the tolerogen can be aligand, such as a protein on the surface of a neoplastic cell, or agrowth factor, such as a protein associated with angiogenesis, or aviral receptor, such as the chemokine receptor CCR5, and cytokines, suchas TNF-α. Fragments of these “full-length” tolerogens are also desirablefor some embodiments. That is, in some embodiments the tolerogen can bean entire molecule (e.g., full-length) but most often, the tolerogencomprises only a portion or fragment of the full-length molecule (e.g.,partial-length). Desirable tolerogens comprise at least 5 to 500consecutive amino acids of the full-length molecule, advantageously 5 to200, and preferably 5 to 50. Preferably, the tolerogen and the viralcapsid protein are derived from different organisms, viruses, orinfectious agents. Other embodiments include an isolated complexcomprising one of the compositions described above joined to a cell ofthe immune system (e.g., a B cell, a T cell, or a dendritic cell) and apharmaceutical comprising one of the compositions described above.

[0024] The compositions, isolated complexes, and pharmaceuticals of theinvention are used as biological tools, therapeutics, and prophylacticsfor the study of B cell tolerance, identification of agents thatgenerate auto-antibodies, and treatment and prevention of humandiseases, such as viral infection, chronic inflammation, and cancer. Inone embodiment, for example, a method to identify agents that generateautoantibodies is provided. By this approach, a composition of theinvention is provided to a subject, antibodies are then isolated fromthe subject, and a determination of whether the isolated antibodiesinteract with the tolerogen presented by the composition is made.Subsequently, the immunogen is identified as one that breaks B celltolerance by the ability of the isolated antibodies to interact with thetolerogen. In another embodiment, a method of generating antibodies to atolerogen is provided in which a subject in need of antibodies to atolerogen is identified and then is provided a therapeuticallybeneficial amount of a composition of the invention. Additionally,methods of treatment and prevention of HIV infection, chronic viralinfection, cancer and inflammation are provided, which involve the stepof providing a pharmaceutical comprising a composition of the invention.For example, breast cancer and rheumatoid arthritis can be treated byinducing the production of antibodies directed against ErbB-2 and TNF-α,respectively. (Maini, R. N. et al., Imm. Reviews, 144:195-223 (1995);Baselga, J. et al., J. Clin. Oncol., 14:737-44 (1996)). Further,polyclonal and monoclonal antibodies directed to epitopes on thechimeric and conjugated VLPs of the invention are embodiments.

[0025] Evidence of our discovery is provided in two groups ofexperiments provided below. In a first exemplary demonstration, B celltolerance to the mouse chemokine receptor (mCCR5) central antigen wasabrogated by immunizing mice with chimeric VLPs having the mCCR5tolerogen. In these experiments, a peptide representing an extracellularloop of the mouse chemokine receptor CCR5 was incorporated into aneutralizing epitope of the bovine papillomavirus virus L1 coat protein(BPV-1) by conventional cloning techniques. L1 has the intrinsiccapacity to self-assemble into virus-like particles (VLPs) that inducehigh levels of neutralizing antibodies, even without adjuvant.(Kirnbauer, R. et al., Proc. Natl. Acad. Sci., USA, 89:12180-12184(1992); Greenstone, H. L. et al., Proc. Natl. Acad. Sci. USA,95:1800-1805 (1998)). The CCR5 receptor is expressed in numerous celltypes and tissues, including memory T cells and macrophages. (Zhang, L.et al., J. Virol., 72:5035-5045 (1998)) Recombinantly produced chimericproteins called “L1-CCR5” self-assembled into particulate structureshaving an ordered array of capsomeres (hereafter designated asvirus-like particles or VLPs) that were used as immunogens. Those havingordinary skill in the art will appreciate that CCR5 is known to be theco-receptor for M-tropic strains of HIV, and that monoclonal antibodiesto human CCR5 block HIV infection of human cells in vitro.

[0026] As detailed below, mice administered with the L1-CCR5 immunogenproduced auto-antibodies that bound to native mouse CCR5, inhibitedbinding of the RANTES ligand, and blocked HIV-1 infection of anindicator cell line that expressed a human-mouse CCR5 chimera. We alsoshow that the long-term effects of the treatment protocol on mice wereminimal. Further, we demonstrate that auto-antibodies to CCR5 can beproduced in primates. These experiments provide evidence that B celltolerance to a cell surface self antigen that has co-evolved with animmune system can be broken. These novel compositions can beincorporated into pharmaceuticals and can be used to treat and/orprevent HIV infection.

[0027] In a second group of experiments, we provide evidence that theproduction of autoantibodies to Tumor Necrosis Factor-α (TNF-α) can beinduced by inoculating a subject with conjugated VLP comprising afragment of TNF-α. The immunogen was created by joining astreptavidin/TNF-α fusion protein (SA-TNF-α) to biotinylated L1-VLPs.Mice inoculated with SA-TNF-α VLP conjugates produced auto-antibodiesthat neutralized the effects of TNF-α on a TNF-α sensitive cell line(L929 cells). These novel compositions can be incorporated intopharmaceuticals and can be used to treat and/or prevent chronicinflammatory disease and other diseases associated with excessiverelease of TNF-α including, but not limited to, rheumatoid arthritis,Crohn's disease, ulcerative colitis, cancer, disseminated sclerosis,diabetes, psoriasis, osteoporosis, and asthma. In the disclosure belowand the examples that follow, we discuss these two groups of experimentsin greater detail.

[0028] A Chimeric VLP that Breaks Immune Tolerance and Inhibits HIVInfection

[0029] While investigating whether auto-antibodies against a selfantigen can be induced, we discovered that B cell tolerance can beabrogated by placing the antigen in a context that mimics the orderedsurface of a viral particle. In our initial experiments, we inserted themouse chemokine receptor mCCR5 into an immunodominant site of the bovinepapillomavirus L1 coat protein. The recombinant protein was called“L1-CCR5”, which is a self-assembling chimeric L1 protein that includesa plurality of amino acids encoding a CCR5 epitope. Papillomaviruseswere selected because they are highly specific immunogens. Eachvertebrate species is infected by a distinct group of papillomaviruses,with each group comprising several papillomavirus types. Neutralizingantibodies against the virions of one papillomavirus type do notordinarily confer immunity against another type.

[0030] Papillomaviruses are examples of non-enveloped viruses thatreplicate in the epithelia of a wide variety of animal species to resultin the formation of benign epithelial and fibro-epithelial tumors orwarts. Papillomavirus particles are about 55 nm in diameter andencapsidate an approximately 8 kb double-stranded DNA genome containedin a nucleohistone core (Baker et al., Biophys J., 60:1445 (1991)). Thecapsids are composed of two virally encoded proteins, L1 and L2, thatmigrate on SDS-PAGE gels at approximately 55 kDa and 75 kDa,respectively (Mose Larson et al., J. Virol., 61:3596 (1987)). The L1major capsid protein is arranged in 72 pentamers which associate withT=7 icosahedral symmetry. There are approximately 12 L2 capsid proteinsper virion. (Baker et al., Biophys J., 60:1445 (1991)).

[0031] The L1 protein has the capacity to self-assemble so that largeamounts of virus-like particles (VLPs) can be generated by expression ofthe L1 protein from a given papillomavirus in a variety of recombinantexpression systems. (Kirnbauer et al., Proc. Natl. Acad. Sci. USA,89:12180 (1992) (BPV-1, baculovirus expression system); Hagensee et al.,J. Virol., 67:315 (1993) (HPV-1, vaccinia virus expression system);Kirnbauer et al., J. Virol, 67:6929 (1993) (HPV-16, baculovirusexpression system); Rose et al., J. Virol., 67:1936 (1993) (HPV-11,baculovirus expression system); Sasagawa et al., Virol., 206:126 (1995)(HPV-16, yeast expression system); Nardinelli-Haefliger et al.,Infection and Immunity, 65:3328 (1997) (HPV-16, bacterial expressionsystem)). Although not required for assembly, L2 is incorporated intoVLPs when co-expressed with L1 (L1/L2 VLPs) in cells.

[0032] Immunization of rabbits with native virions or L1 VLPs, but notwith denatured L1 proteins, induces high titers of neutralizing serumantibodies (Christensen et al., J Virol., 64:3151 (1990); Kirnbauer etal., Proc. Natl. Acad. Sci. USA, 89:12180 (1992); Pilacinski et al.,Bio/Technology, 2:356 (1984); Segre et al., Am. J. Vet. Res., 16:517(1955)). The polyclonal and monoclonal neutralizing antibodies generatedagainst native particles recognize conformationally dependent epitopes(Christensen et al., Virus Res, 28:195 (1993); Christensen et al.,Virology, 181:572 (1991)). Although, the nature of the humoral immuneresponse against papillomavirus antigens is well established, no oneappreciated or expected that the ordered geometry of L1 VLPs could beexploited to present a tolerogen to the immune system in a manner thatpromotes a potent immune response and, thus, breaks B cell tolerance.

[0033] Generation of chimeric L1-CCR5 particles required inserting theCCR5 peptide into a region of L1 that would not disrupt the ability ofL1 to form particles. (See Example 1). Although the precise structurallocation and function of most L1 amino acids are not known, amino acidchanges that disrupt the neutralizing epitopes of various humanpapillomaviruses without affecting capsid assembly have been mapped tothree non-contiguous regions of L1. (Ludmerer, S. W., Benincasa, D. &Mark III, G. E., J. Virol., 70:4791-4794 (1996); Ludmerer, S. W. et al.,J. Virol., 71:3834-3839 (1997); Roden, R. B. et al., J. Virol.,71:6247-6252 (1997)). As it was likely that amino acids at these siteswere on the surface of the capsid, the analogous sites in BPV-1 L1 weretargeted for peptide insertion. Therefore, three L1-CCR5 chimeras wereconstructed in which the L1 sequence at BPV-1 L1 amino acids 130-136,275-285, or 344-350 was replaced with a sequence predicted to encode a16 amino acid peptide corresponding to the first EC loop of mouse CCR5(mCCR5) from C57B1/6 (B6) mice. These chimeras were designated L1-CCR5chimeras 1, 2, and 3, respectively.

[0034] Recombinant baculoviruses containing L1-CCR5 chimeras weregenerated, and the resulting L1-CCR5 particles were purified by gradientcentrifugation. (Kirnbauer, R. et al., Proc. Natl. Acad. Sci. USA,89:12180-12184 (1992)). To determine if the chimeric L1-CCR5 moleculesassembled into VLPs, capsomeres, or other particulate forms, Superose 6gel filtration chromatography was performed on preparations of the threeL1-CCR5 chimera. (See Example 2). Only preparations of L1-CCR5 chimera 1eluted in a fraction indicating an assembled particulate structure.Therefore, further analysis was limited to this chimera. Examination ofchimera 1 particles by electron microscopy revealed many particles whichwere smaller than wild type L1 VLPs, approximately 28 nm vs 55 nm.Morphologically, the L1-CCR5 chimeric particles resembled polyomavirus12 ICOSA shells (T=1 particles), which are composed of a regular arrayof 12 pentameric capsomers of the polyomavirus major coat protein VP1,and can be generated upon in vitro reassembly of VP1 capsomeres at highionic strength. (Salunke, D., Caspar, D. L. D. & Garcea, R. L.,Biophysical Journal, 56:887-900 (1989)). Small particles of a similarsize to the L1-CCR5 particles are often found as a minor component ofwild type BPV-1 L1 VLP preparations.

[0035] To examine whether the CCR5 chimeric particles could induceanti-CCR5 antibodies, C57B1/6 mice (a strain which encodes the identicalCCR5 sequence as the insert sequence) were vaccinated with L1-CCR5particles, denatured L1-CCR5 protein, or wild type VLPs. (See Example3). Sera from these mice were tested for reactivity to CCR5 peptide andwild type VLPs by ELISA (FIG. 1A). Sera from control mice inoculatedwith wild type VLPs had no anti-CCR5 ELISA reactivity, but inoculationwith L1-CCR5 particles induced sera with high anti-CCR5 ELISA titers.These titers ranged from 3×10³ to 3×10⁴ in the three animals inoculatedin combination with Freund's adjuvant, and measured 3×10³ in the twoanimals inoculated without adjuvant. In contrast, noCCR5-peptide-specific antibodies were detected in mice inoculated withdenatured L1-CCR5 particles in combination with adjuvant. The lack ofreactivity of the denatured L1-CCR5 particles was limited to the CCR5peptide, since the denatured material elicited high titers of anti-L1antibodies (FIG. 1B).

[0036] While these results provided evidence that the L1-CCR5 particleselicit antibodies to the CCR5 peptide, the possibility existed thatthese antibodies might not recognize the peptide in its nativeconformation as part of membrane associated mCCR5. To eliminate thispossibility, an experiment was performed in which the ability ofanti-CCR5 antibodies to bind to mCCR5 on cells was tested by flowcytometric (FACS) analysis. (See Example 4). The binding of L1-CCR5particle sera to mCCR5 expressed on primary mouse T cells andmacrophages could not be assessed because of high levels of non-specificmouse IgG binding to these cells. Alternatively, cloned mCCR5 from B6mice was transiently expressed in HeLa-MAGI cells by transfection, andthe binding of purified mouse IgG was measured relative to vectortransfected cells (FIGS. 2A-2G). By this assay, IgG from L1-CCR5immunized mice bound specifically to the mCCR5 transfected cells (FIG.2A), whereas there was no significant binding with purified IgG fromwild type BPV VLP sera (FIG. 2B), or with a monoclonal antibody (mAB182)that binds to the second EC loop of human (h) CCR5 (FIG. 2C). As acontrol for antibody specificity, mice were inoculated with mCCR5peptide coupled to keyhole limpet hemocyanin (KLH). While these micegenerated an anti-CCR5 peptide antibody response, with ELISA titers of10⁵ against CCR5 peptide coupled to bovine serum albumin (BSA), the IgGpurified from the sera of these mice failed to bind mCCR5 expressingcells (FIG. 2D). Thus, the L1-CCR5 induced antibodies, in contrast tothose induced by the KLH-coupled peptide, function as trueauto-antibodies, in that they bind native mCCR5.

[0037] As another approach to examine the ability of the antibodies tobind native mCCR5, we examined whether the L1-CCR5 sera could competewith a chemokine ligand for mCCR5 for binding to HeLa-MAGI cellstransiently transfected with mCCR5 (FIG. 3). (See Example 5). The mousechemokines MIP-1α, MIP-1β, and RANTES are ligands for mCCR5. Inaddition, the human homologs of MIP-1β and RANTES are able to bind tomCCR5. (Meyer, A. et al., J. Biol. Chem., 271:14445-14451 (1996); Nibbs,R. J. B. et al., J. Biol. Chem., 272:12495-12504 (1997)). In thecompetition assay, commercially available iodinated human RANTES wasused. A 1:30 dilution of L1-CCR5 sera displaced approximately 66% of theiodinated human RANTES (similar to the displacement observed using a100-fold excess of cold RANTES), compared with 37% displacement with a1:30 dilution of wild type L1 VLP sera. The 1:75 and 1:150 dilutions ofL1-CCR5 sera displaced 25% and 17% of the iodinated RANTES,respectively, whereas no significant displacement was observed usingcontrol sera at these dilutions. Previous studies have shown thatMIP-1α, MIP-1β, and RANTES bind to the to 2nd EC loop of hCCR5, sincetheir binding was blocked by monoclonal antibody to this loop but not byantibody to the amino terminus of hCCR5. (Wu, L. et al, J. Exp. Med.,186:1373-1381 (1997)). Our results from these experiments providesevidence that antibodies binding to the first EC loop of mCCR5, which islocated between these two sites, can partially block RANTES binding,perhaps because of the proximity of this loop to the 2nd EC loop.

[0038] The ability of L1-CCR5 induced antibodies to block M-tropic HIV-1infection was also tested. (See Example 6). The interaction betweenHIV-1 envelope and hCCR5 is complex, likely strain dependent, andprobably involves several EC regions of CCR5. Specifically, monoclonalantibody studies have implicated the 2nd EC loop and the NH2-terminalregion of hCCR5, and studies of chimeric receptors have indicated thatthe first and third EC loops of hCCR5 also contribute to its interactionwith HIV-1. (Wu, L. et al., J. Exp. Med., 186:1373-1381 (1997); Rucker,J. et al., Cell, 87:437-446 (1996); Atchison, R. E. et al., Science,274:1924-1926 (1996); Alkhatib, G. et al., J. Biol. Chem.,272:19771-19776 (1997); Picard, L. et al., J. Virol., 71:5003-5011(1997); Ross, T. M., Bieniasz, P. D. & Cullen, B. R., J. Virol.,72:1918-1924 (1998)). Although mCCR5 does not function as an HIV-1coreceptor, a human-mouse chimeric receptor (HMHH), which contains thefirst EC loop of mCCR5 (the B6 mouse sequence) in a background of hCCR5,has coreceptor activity (albeit at low efficiency) when expressed inhuman cell lines. (Kuhmann, S. E. et al., J. Virol., 71:8642-8656(1997)). We used this chimeric receptor to test whether L1-CCR5 seracould block M-tropic HIV-1 infection. To confirm that IgG purified fromL1-CCR5 sera would bind HMHH, FACS analysis was performed on HeLa-MAGIcells transiently transfected with HMHH. Positive binding was obtainedwith IgG from L1-CCR5 mice and with a positive control monoclonalantibody that binds to the 2nd EC loop of human CCR5, while IgG fromwild type L1 VLP mice did not bind HMHH (FIGS. 2E-2G).

[0039] Based on these results, sera from L1-CCR5 mice were tested fortheir ability to inhibit the infection of the M-tropic BaL strain ofHIV-1, in a single replication cycle assay, using the MAGI indicatorcell line. (Kimpton, J. & Emerman, M., J. Virol., 66:2232-2239 (1992)).When indicator cells transiently transfected with HMHH were infectedwith HIV-1 BaL in the presence of L1-CCR5 sera, dilutions of 1:15, 1:30,and 1:75 exhibited 65%, 50%, and 45% neutralization, respectively, ofinfectivity (FIG. 4). At the same dilutions, control sera from wild typeL1 VLP mice exhibited some non-specific neutralization, but it was only25% at the 1:15 dilution and 15% at 1:30 and 1:75. In comparison,indicator cells infected with HIV-1 BaL in the presence of dilutions ofhCCR5 binding monoclonal antibody (mAB 182) (at an initial concentrationof 1 μg/μl) used as a positive control exhibited a similarneutralization curve (FIG. 5). The L1-CCR5 sera were also tested forneutralization activity against the T-cell tropic isolate HIV-1 LAI,and, as expected, failed to show any neutralization above backgroundlevels against this isolate.

[0040] One concern of auto-antibody induction is that such proceduresmay have deleterious long-term consequences for the immunized animal,possibly including uncontrolled antigenic stimulation from the nativeCCR5 protein. However, in three mice that were monitored over a sixmonth period after L1-CCR5 particle inoculation, we observed a two- toeight-fold decrease in the titer of CCR5-specific antibodies over thisperiod; this decline was roughly equivalent to a parallel decline in thetiter of L1-specific antibodies. Two of the animals exhibited two-folddeclines in anti-CCR5 antibody titers and three-fold declines in anti-L1antibody titers. The third animal exhibited an eight-fold decline in itsanti-CCR5 titer and a ten-fold decline in its anti-L1 antibody titer.These results provide evidence that continued exposure to native CCR5does not lead to continuous B cell induction, presumably because thecellular protein remains in a context that is ignored by the immunesystem, and, moreover, because the anti-CCR5 response dependsexclusively on exposure to the CCR5 peptide on L1-CCR5 particles. Theimmunized mice maintained the same weight as control mice, and autopsiesperformed on two of the mice six months after the final boost did notreveal any gross pathological changes.

[0041] In humans, CCR5 is expressed predominantly on memory T cells(CD3⁺, CD4⁺, CD26^(hi)). Additionally, from 1 to 10% of macrophages inthe thymus, spleen and lymph nodes express CCR5. (Zhang, L. et al., J.Virol., 72:5035-5045 (1998)). FACS analysis of mouse mononuclear cellsfrom spleen, thymus, and peripheral blood indicated that there was nodecline in spleen or peripheral blood macrophage and T cell subsets thatexpress CCR5 compared to control mice. Thus, according to our analysis,the mice immunized with L1-CCR5 particles did not suffer grosspathological changes over the period of observation.

[0042] More evidence supporting the benefits of using chimeric VLPs forbreaking B cell tolerance in humans and specifically for the treatmentand prevention of HIV infection, was obtained from primate studies inwhich auto-antibodies to a macaque CCR5 polypeptide were produced. (SeeExample 7). In these experiments, a recombinant expression constructencoding a macaque L1-CCR5 fusion protein was made. We cloned thecorresponding human/macaque CCR5 peptide (they are the same) into theidentical place in the L1 major capsid protein as used in the mouseexperiments. Next we assessed capsid particle self assembly. Whencompared to our previous experiments with mouse L1-CCR5, particleformation was inefficient with the chimeric macaque L1-CCR5.Nevertheless, we were able to purify sufficient particles to immunizepig tail macaques. Four of the five animals that were vaccinated threetimes with the preparation, in the presence of Titer Max adjuvant,clearly produced CCR5 specific antibodies as measured in an ELISA assay(FIG. 5). We believe that a better VLP-based immunogen for generatingauto-antibodies to human/macaque CCR5 can be generated by findinganother site for insertion of the CCR5 peptide that would both displaythe foreign peptide on the VLP surface and would be more compatible withself assembly. Additionally, as described below, we believe thatconjugated VLPs having the human/macaque CCR5 tolerogen will induce abetter immune response.

[0043] The results from the first group of experiments demonstrate thatincorporation of a peptide from the EC portion of a central antigen,mCCR5, into the regular array of a papillomavirus particle, followed byimmunization of these particles, can induce auto-antibodies that bind tothe receptor and block ligand and HIV-1 binding. Auto-antibodies tomCCR5 declined over time at a rate that was similar to the decline in L1specific antibodies, suggesting that B cell stimulation by endogenouscell-surface CCR5 was not induced.

[0044] The anti-self antibodies induced by L1-CCR5 particles efficientlybound mCCR5 expressed on the cell surface, indicating that they functionas true auto-antibodies. In contrast, antibodies induced by KLH-coupledCCR5 peptide failed to bind to native mCCR5. It is likely that bindingauto-antibodies do not just recognize this particular amino acidsequence, but the tolerogen sequence in its native conformation.Moreover, the IgG from L1-CCR5 immunized mice block binding of a CCR5ligand and inhibit HIV-1 infection via a chimeric CCR5 protein thatcontains the mouse CCR5 peptide, further demonstrating the specificityof these auto-antibodies and the therapeutic usefulness of aspects ofthe invention. The inhibition observed in these assays, was consistent,reproducible, specific, and similar to a control monoclonal antibodyagainst the second EC loop of human CCR5.

[0045] The first EC loop of mCCR5 was chosen for our initialinvestigations because it allowed us to simultaneously test our approachto breaking B cell tolerance and provide a novel method to induce thebody of a subject to inhibit HIV-1 infection. Because HIV-1 infectedindividuals who are heterozygous for an inactive CCR5 allele havedelayed progression to AIDS, even partial reduction in CCR5 expressioncan have clinically significant effects. (Liu, R. et al., Cell,86:367-77 (1996); Samson, M. et al., Nature (London), 382:722-5 (1996);Winkler, C. et al., Science, 279:389-93 (1998)). Our results alsodemonstrate that primates have the capacity to produce antibodiesspecific for CCR5 provided that the antigen is presented in anappropriate immunogen.

[0046] We observed no adverse effects of auto-antibody induction in micethat were followed for six months from the initial inoculation. While wedid not test for auto-reactive T cells, we would not expect to break Tcell tolerance to CCR5. T cells that recognize central auto-antigens arestrongly selected against during the development of the immune system.Presumably the T cell help needed for immunoglobulin class switching toproduce anti-CCR5 IgG is directed against the linked viral protein.Conversely, in adult animals there is a continuous generation ofantibodies with new specificities as a result of RAG reactivation andperipheral editing of B cell receptor genes. (Han, S. et al., Science,278:301-5 (1998); Papavasiliou, F. et al., Science, 278:298-301 (1998);Hertz, M. et al., Nature, 394:292-5 (1998)).

[0047] A Conjugated VLP That Breaks Immune Tolerance and Inhibits TNF-αActivity

[0048] Our second approach to break B cell tolerance involves the use ofconjugated VLPs constructed by the addition of tolerogens to the outersurface of pre-formed VLPs. Once assembled, VLPs and capsomericstructures are quite stable and the addition of large or smalltolerogens can be easily accomplished. In contrast to the chimeric VLPapproach, wherein the size of the tolerogen is usually small because ofthe perturbation of self assembly, the conjugated VLP approach allowsfor the assembly of larger tolerogens to the VLP, which would providemore antibody epitopes and thus a more diverse set of auto antibodies.This strategy might also be applicable to polypeptides of more variedsequence and size than would genetic insertion of the sequences into themajor capsid protein.

[0049] In our first experiments using the conjugated VLP approach, wejoined the tolerogen TNF-α to VLPs by way of a biotin-streptavidininteraction. L1 VLPs were shown to be heavily biotinylated usingsulfo-NHS-biotin (Pierce), which biotinylates exposed lysine residues.Under saturating conditions, the biotinylated VLPs bound streptavidin ata ratio of approximately three streptavidin tetramers per one L1molecule. This result provided evidence that a streptavidin/tolerogenfusion protein could be presented to the immune system as a dense,ordered, and closely packed array of epitopes.

[0050] Accordingly, in E. coli, we generated a fusion protein comprisingthe streptavidin core linked to a twenty amino acid long fragment ofmouse TNF-α, referred to as SA-TNF-α. The fusion protein was purified asinsoluble inclusion bodies, solubilized in guanidine-HCl, and refoldedby dialysis into physiologic buffer. The refolded fusion protein wasthen bound to biotinylated L1 VLPs, prepared as described above, so asto create the SA-TNF-α/VLP immunogen. The SA-TNF-α fusion protein boundto biotinylated VLPs with high occupancy (FIG. 6). Next, theSA-TNF-α/VLP immunogen was injected into mice so as to elicit anautoantibody response. Two injections of the conjugated VLPs into mice(3×5 μg) induced high titers of antibodies that bound native mouse TNF-αin an ELISA (See Table 1). In contrast, immunization with eitherSA-TNF-α alone or VLP alone failed to elicit a consistent or highautoantibody response. (See Tables 2 and 3). After a third injection ofSA-TNF-α/VLP, titers of TNF-α antibodies reached 10⁵ in all mice. TABLE1 Mouse titers after two immunizations with SA-TNFα protein coupled tobiotinylated VLPs SA-TNF-α/VLP + Adjuvant − Adjuvant 10⁴ 10⁴ 10⁴ 40 160160

[0051] TABLE 2 Mouse titers after two immunizations with SA-TNFa proteinalone SA-TNTα + Adjuvant − Adjuvant 10² <10 <10 10 <10 <10

[0052] TABLE 3 Mouse titers after two immunizations with SA-TNFα proteinalone VLPs + Adjuvant − Adjuvant <10 <10 <10

[0053] These results established that the SA-TNF-α/VLP immunogenefficiently broke B cell tolerance. More evidence that the conjugatedVLP effectively broke B cell tolerance was obtained from TNF-αcytotoxicity assays. In these experiments, the ability of theSA-TNF-α/VLP immunogen to prevent TNF-α mediated apoptosis of anindicator cell line (L929 cells) was determined. Sera obtained from miceinnoculated with either streptavidin bound VLPs (controls) orSA-TNF-α/VLPs was incubated with TNF-α and the sera treated or non-seratreated TNF-α samples were added to cells in culture. The ability of thesera to neutralize TNF-α activity was measured by an increase in thepercentage of surviving cells. Sera from the SA-TNF-α/VLP innoculatedmice (at a 5% solution) lead to a three-fold increase in cell survivalover background levels (FIG. 7). Given the encouraging results with themouse TNF-α fusion proteins, we made similar streptavidin fusionproteins for each of the four extracellular domains of macaque CCR5. Allfour fusion proteins have been generated and purified from E. coliinclusion bodies. We have been able to refold three of the four suchthat they strongly bind biotinylated VLPs.

[0054] As shown in the discussion above and the following examples, apeptide sequence corresponding to a tolerogen can abrogate B celltolerance to the native protein, when presented in the context of ahighly organized array of assembled chimeric or conjugated viralcapsomeres. The ability to abrogate B cell tolerance using theprocedures described herein has numerous applications. For example, thistechnique can be used to generate mouse anti-self monoclonal antibodies.Additionally, this approach is effective as a means of modulating theactivity of a soluble protein in order to examine its function in normalor disease processes in experimental animal models. Moreover, inductionof auto-antibodies provides an effective alternative to monoclonalantibody therapy for human disease, such as in the treatment of breastcancer and rheumatoid arthritis with antibodies directed against ErbB-2and TNF-α, respectively (Maini et al., Immunol. Rev., 144:195 (1995);Baselga et al., J. Clin. Oncol., 14:737 (1996)). The discussion belowdescribes more aspects that concern embodiments of the invention.

[0055] Supports and Capsomeric Structures

[0056] While virus-like particles or capsomeric structures represent apreferred system for delivering self peptides to the immune system tostimulate production of auto-antibodies, we also intend for theinvention to embrace other structured assemblages that can present atolerogen in an ordered, closely spaced repetitive array. These supportshave an ordered assembly of subunits and allow for at least one B cellepitope of a tolerogen to be joined to the support in a regular,repetitive array. Preferably, the supports and capsomeric structures arecapable of presenting antigen with spacing of about 10-500 angstroms,advantageously about 50-300 angstroms, and preferably about 100angstroms. That is, spacing between presented antigen molecules can begreater than or equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163,164,165,166,167,168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 210, 201, 202, 203, 204, 205,206,207, 208, 209,210, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432,433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 angstroms.Modified T-independent type 2 antigens (TI-2) may behave like VLPs inthis regard. These include pneumococcal polysaccharide, Salmonellapolymerized flagellin, dextran and hapten-conjugated ficol(polysucrose).

[0057] Although virus-like particles of papillomavirus have beenemployed in the exemplary demonstration presented herein, virus-likeparticles of other papillomaviruses and non-papillomaviruses also arecontemplated for use in stimulating production of autoantibodies.Infectious virus are also envisioned. Attenuated or inherentlynon-pathogenic viruses can be modified in a similar fashion and used togenerate autoantibodies. Examples of chimeric VLPs particularlycontemplated for use in connection with the invention are thosedescribed in Intervirology, 39:1 (1996), herein incorporated byreference. Among the chimeric VLPs contemplated for use in stimulatingproduction of autoantibodies are: BPV-1, HPV-1, HPV-6, HPV-11, HPV-16,HPV-18, HPV-33, HPV-45, CRPV, and COPV. Also contemplated are: B19parvovirus, Hepatitis B virus core particles, Hepatitis B surfaceantigen particles, HIV gag particles, Tobacco Mosaic Virus, Cowpeamosaic virus, Yeast Ty particles, and RNA phage. Virus-like particleshave been made, and chimeric VLPs can be made for SV40, Polyomavirus,Adenovirus, Herpes Simplex, Rotavirus, and Norwalk Virus. Notably,workers in the art have already determined the complete nucleotidesequence of the entire genomes of many papillomavirus, including: BPV-1,BPV-2, BPV-4, CRPV, DPV, EPV, HPV-1, HPV-5, HPV-6, HPV-8, HPV-11,HPV-16, HPV-18, HPV-31, HPV-33 and RhPV. Preferred capsid proteins thatare used to construct the capsomeric structures of embodiments of theinvention, however, include proteins from icosohedral viruses or virusesthat have a natural mammalian reservoir host. Thus, polynucleotidesequences encoding many different major and minor coat proteins that canbe used in connection with the methods described herein already areknown.

[0058] It is to be understood that VLPs that include major and/or minorcoat proteins can be used to prepare immunogenic compositions accordingto the methods disclosed herein. In the particular case of thepapillomavirus L2 minor coat protein, it is to be understood that L2chimeras may expose the inserted antigen on the surface. The targetantigens are external. An L2/E7 fusion that we have used to generatepapillomavirus E7 antibodies, when incorporated in VLPs, has the first110 amino acids of BPV L2 fused to the entire HPV16 E7 polypeptidesequence. (Lowy et al., U.S. Pat. No. 5,618,536 herein incorporated byreference.) In this instance, the E7 sequence was fused to a site of L2that had previously been shown to correspond to a virus-neutralizingepitope (Roden, et al., J. Virol., 68:7570 (1994)). In the disclosurebelow, several approaches to join tolerogens to the ordered assemblagesof the invention are provided.

[0059] Approaches to Make Supports or Capsomeric Structures that PresentTolerogens

[0060] As discussed above, in general, two different approaches can beemployed to incorporate tolerogens into the structures of virus-likeparticles. By one approach, there first is created a genetic constructthat encodes an amino acid sequence including both virus coat proteinsequences and the self peptide sequence of interest. The resultingconstruct encodes a single chimeric polypeptide that displays the selfpeptide on an outer surface of a particle following self-assembly of thehybrid coat protein to form capsomeric structures or VLPs. According tothe second approach, the self peptide displayed on the outer surface ofthe VLP is linked directly or indirectly to a plurality of subunitproteins that comprise a preformed VLP. For example, wild typepapillomavirus L1 protein can be a recombinant coat protein coupled to afirst binding agent having an association constant for a second bindingagent ranging from 10⁷-10¹⁰, from 10⁴-10⁸, from 10¹⁰-10¹², or from10¹²-10¹⁶. The second binding agent can be adapted for coupling to theself peptide. In a particularly preferred embodiment of the invention,biotinylated wild type VLPs are first produced. This can be accomplishedby biotinylating pre-formed VLPs. Next, the biotinylated VLPs arecombined with an avidin- or streptavidin-linked self polypeptide to formcomplexes having surfaces whereon the self peptide is displayed. In thisfashion multiple copies of the self peptide are indirectly coupled tothe VLP such that the self peptide is not integrated into the peptidebackbone of the coat protein. Thus, compositions that include a hybridcoat protein linked to a self peptide, either as an integral part of thehybrid coat protein polypeptide sequence or indirectly, such as througha biotin linkage, are intended to fall within the scope of theinvention.

[0061] Embodiments of the invention desirably provide tolerogens in sucha form or in such a way that a sufficient affinity, abrogation of B celltolerance or inhibition of a disease state (e.g., viral infection,neoplasis, or inflammation) is achieved. While a natural monomerictolerogen (that is, a tolerogen that presents a discrete molecule, thus,carrying only a small number of epitopes) can be sufficient to achieve adesired response, a synthetic tolerogen or a multimeric immunogen (e.g.,a VLP presenting multiple molecules of the tolerogen, thus, having agreater number of the same epitopes) often times can elicit a greaterimmune response. It should be noted that the term “multimeric” refers tothe presence of more than one identical molecule on a support orcapsomeric structure. For example, several identical molecules of CCR5or fragments thereof displayed on a VLP. The term multimeric should bedistinguished from the term “multimerized”, which refers to a support orcapsomeric structure joined to hybrid molecules, wherein each hybridmolecule comprises multiple copies of the tolerogen or individualepitopes thereof joined in tandem. For example, each individualmultimerized tolerogen can comprise a twenty amino acid long fragment ofCCR5 that is randomly repeated with or without interspersed linkers(e.g., 8 phage linkers) and a plurality of multimeric tolerogens can bejoined to a support or capsomeric structure so as to form amultimerized/multimeric immunogen.

[0062] A multimeric immunogen (synthetic or natural) that effectivelybreaks B cell tolerance can be obtained by joining tolerogens to asupport or a capsomeric structure. Supports suitable for this purposeinclude, but are not limited to, polyacrylamide beads, agarose beads,polystyrene beads, magnetic beads, latex particles, carbohydrateassemblies (e.g., oligosaccaride-based beads or assemblies), lipidassemblies (e.g., lipid membranes), protein assemblies or polymers(e.g., poly-L-lysine or poly-D, L-alanine) and other supports known inthe art to have an organized, symmetrical assembly of subunits.Inorganic carriers, such as silicon oxide material (e.g. silica gel,zeolite, diatomaceous earth or aminated glass) to which the tolerogen iscovalently linked through a hydroxy, carboxy or amino group and areactive group on the carrier can also be used with some embodiments.

[0063] In several embodiments, the tolerogen is joined to the support orcapsomeric structure by way of a linker, which can be a bond between twochemically reactive species, a ligand/receptor interaction, or a peptidethat has been joined to the tolerogen so as to allow for attachment tothe support or capsomeric structure or to provide greater freedom ofassociation of the tolerogen with a cell of the immune system. In someembodiments, for example, the support or capsomeric structure has ahydrophobic surface that interacts with a portion of the tolerogen by ahydrophobic non-covalent interaction. In some cases, the hydrophobicsurface of the support is a polymer such as plastic or any other polymerin which hydrophobic groups have been linked such as polystyrene,polyethylene or polyvinyl.

[0064] Additionally, the tolerogen can be covalently bound to a supportor capsomeric structure including proteins and oligo/polysaccarides(e.g. cellulose, starch, glycogen, chitosane or aminated sepharose). Inthese later embodiments, a reactive group on a tolerogen, such as ahydroxy or an amino group, is used to join to a reactive group on thesupport or capsomeric structure so as to create the covalent bond.Embodiments can also comprise a support with a charged surface thatinteracts with the tolerogen. Additional embodiments comprise a supportthat has other reactive groups that are chemically activated so as toattach a tolerogen. For example, cyanogen bromide activated matrices,epoxy activated matrices, thio and thiopropyl gels, nitrophenylchloroformate and N-hydroxy succinimide chlorformate linkages, oroxirane acrylic supports are used. (Sigma).

[0065] In other embodiments, the interaction of biotin with avidin-likemolecules (e.g., streptavidin and neutraavidin) is exploited. Aspresented earlier, VLPs can be biotinylated and can be easily joined totolerogen/streptavidin fusion proteins. By inserting more lysine orcysteine molecules in capsid proteins, greater biotinylation can beachieved and, thus, more tolerogen can be added to the surface of a VLP.Further, by using site-specific mutagenesis techniques, lysine orcysteine molecules can be strategically inserted so as to establish aVLP having a dense and highly organized repetitive array of tolerogens.As one of skill will immediately appreciate, the converse also can beperformed, that is, the use of streptavidin/capsid protein fusions andbiotinylated tolerogens. In another embodiment, 8 linkers of anappropriate length are inserted between the tolerogen and the support orcapsomeric structure so as to encourage greater flexibility and overcomeany steric hindrance that can be present. The determination of anappropriate length of linker that allows for an optimal immune responsecan be made by screening the tolerogen with varying linkers in thevarious assays described herein

[0066] A composite support having more than one type of tolerogen isalso an embodiment. A “composite support” can be a macromolecularstructure used to join or immobilize two or more different tolerogens.The composite supports are also constructed by utilizing hydrophobicinteractions and covalent linkages formed through reactive groups, asdetailed above. Further, linkers, such as 8 linkers, of an appropriatelength between the tolerogens and the support are inserted in someembodiments so as to encourage greater flexibility in the molecule andovercome steric hindrance. The determination of an appropriate length oflinker that allows for an optimal immune response can be made byscreening the iolerogens with varying linkers in the assays detailed inthe present disclosure.

[0067] In other embodiments of the present invention, the multimeric andcomposite supports discussed above have attached multimerized tolerogensso as to create a “multimerized-multimeric support” and a“multimerized-composite support”, respectively. An embodiment of amultimerized tolerogen, for example, is obtained by creating anexpression construct having two or more nucleotide sequences encoding atolerogen joined together by using conventional techniques in molecularbiology. The expressed fusion protein is one embodiment of amultimerized agent and is then joined to a support. A support havingmany such multimerized agents is termed a multimerized-multimericsupport. The multimerized form of a tolerogen can be advantageous formany applications because of the ability to obtain an agent with abetter ability to induce an immune response and, thus, break B celltolerance. The incorporation of linkers or spacers, such as flexible 8linkers, between the protein domains that make-up the multimerized agentcan also be advantageous for some embodiments. The insertion of 8linkers of an appropriate length between protein binding domains, forexample, encourages greater flexibility in the molecule and overcomessteric hindrance between the domains. Similarly, the insertion oflinkers between the multimerized tolerogens and the support encouragesgreater flexibility and reduces steric hindrance presented by thesupport or capsomeric structure. The determination of an appropriatelength of linker that allows for an optimal immune response can beaccomplished by screening the tolerogens with varying linkers in theassays detailed in this disclosure. In a similar fashioncomposite-multimerized-multimeric supports with and without linkers canbe constructed by joining more than one different multimerized tolerogento a support.

[0068] Particularly preferred sites on virus-like particles forinserting self antigens against which an autoimmune response is desiredare virus-neutralizing epitopes. This is because virus-neutralizingepitopes typically are disposed on the surface of the virus and areavailable for antibody binding. These features are desirable forpresenting self antigens to the immune system in the structural contextof a chimeric virus-like particle. Methods for identifyingpapillomavirus neutralizing epitopes have been described by Ludmerer etal., in J. Virol., 70:4791 (1997); Ludmerer et al., in J. Virol.,71:3834 (1997); and by Roden et al., in J. Virol., 71:6247 (1997).Generally, methods for identifying virus-neutralizing epitopes canemploy a monoclonal antibody that preferentially binds to one of twoclosely related L1 proteins and then systematically making recombinantsthat reassort the specific amino acid differences between them.Alternatively, the polypeptide sequences encoding the L1 proteins ofrelated viruses, for example papillomaviruses, can be aligned toidentify segments that are most varied in length. These highly variablepositions likely will be external or internal loops of the capsidprotein. The external loops will be candidates for substitution bypolypeptide sequences of self antigens for which an autoimmune responseis sought. Thus, virus-neutralizing epitopes, as can readily beidentified using routine laboratory procedures, are preferred sites fordisposition of self peptides in the VLPs or capsomeric structures of theinvention. For example, the site of a virus-neutralizing epitope onBPV-1 would be a preferred site on a corresponding VLP for dispositionof a self peptide. In the section below, a discussion of the size oftolerogens that can be used with aspects of the invention is provided.

[0069] Size of Tolerogen on the Support or Capsomeric Structure

[0070] Generally, the number of amino acids representing the antigenthat is incorporated into the structure of the viral coat protein orjoined to the support or capsomeric structure must be large enough tocorrespond to an epitope that is characteristic of the antigen and thatcan fit into the antigen binding site of an antibody. Since it isgenerally accepted that a linear arrangement of 5 to 6 amino acids issufficient to bind to an antigen binding site, it is preferred that atleast 5 amino acids of the self antigen are incorporated into thestructure of the immunogen. However, it is to be understood that agreater number of amino acids can also be used with good results. In theExample presented herein, 16 amino acids of the mouse CCR5 protein wereintroduced into the structure of an L1 major coat protein with goodresults. Longer polypeptide sequences representing tolerogens are alsocontemplated for integration into the structure of the viral coat foruse as immunogens for inducing anti-self immune reactions. Thus, it ispreferred that the immunogenic virus-like particle incorporate selfpolypeptide sequences at least 5 amino acids in length but the length oftolerogen may be greater than 200 amino acids and may include afull-length protein. A desirable range is from 5 to 200 amino acids.That is, the tolerogen can be greater than or equal to 5,6,7, 8,9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178,179,180, 181, 182, 183, 184, 185,186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and200 amino acids in length.

[0071] It is contemplated that biotinylated VLPs will be capable ofcomplexing with, and presenting to the immune system, peptides that aresubstantially longer than 16 amino acids in length. It is also possibleto incorporate into the structure of a hybrid minor coat protein apolypeptide sequence representing a full length protein. Below wedescribe the wide variety of tolerogens that can be presented as anembodiment of the invention.

[0072] Many Types of Tolerogens can be Presented on a Support orCapsomeric Structure

[0073] The invention can be practiced using a wide variety oftolerogens. In preferred embodiments, the tolerogens are self antigens.Preferred self antigens include those for which antibodies targetedagainst that self antigen has been shown to be an effective therapeuticagent. In general, the self antigen will correspond to a self antigen ofthe organism that is immunized with the composition that includes theself antigen linked to the VLP. Thus, the sequence of a human selfpeptide, such as the human CCR5 chemokine receptor, can be introducedinto the structure of a VLP for use in immunizing humans. In this wayhumans can be made to produce autoantibodies. Particular examples ofself antigens that can be used are central self antigens such as TNF-αand CTLA-4.

[0074] TNF-α has been implicated in a number of human diseases, mostnotably as the principle soluble effector in rheumatoid arthritis (RA).(Maini et al., Imm. Reviews, 144:195 (1995)). Rheumatoid arthritis is achronic and painful disease of multiple joints which is thought toaffect around 1% of people worldwide. The symptoms of RA areineffectively treated with drug therapy. This has prompted a desire todevelop alternative therapeutic strategies that target the effectors ofthe disease rather than the symptoms. Anti-TNF-α monoclonal antibodytherapy has produced dramatic improvement in both objective andsubjective measures of RA in human clinical trials (Feldman et al.,Annu. Rev. Immunol., 14:397 (1996)). Unfortunately, the benefits haveproven to be transient and this loss of effectiveness has correlatedwith the development of antibodies against the monoclonal antibody.Coupled with the fact that TNF-α is a small soluble protein that isbiologically active at relatively low serum concentrations, theseobservations make TNF-α an attractive target for an autoantibodyinducing vaccine. Furthermore, there is a good mouse model for TNF-αmediated RA in which to test the concept (Thorbecke et al., Proc. Natl.Acad. Sci. USA, 89:7375 (1992)).

[0075] CTLA-4 is a membrane bound receptor of T cells that appears to bean important regulator of B7 mediated co-stimulation of T cells(Thompson et al., Immunity, 7:445 (1997)). Costimulation is critical forgenerating an effective CD8+ T cell mediated cytotoxic T lymphocyte(CTL) responses. Whether CTLA-4 normally acts to transmit positive ornegative signals after B7 engagement is currently unresolved (Zheng etal., Proc. Natl. Acad. Sci. USA, 95:6284 (1998)). However, antibodies toCTLA-4 clearly potentiate the generation of CTLs in response to tumorantigens in mouse tumor models. Thus, a vaccine for inducingautoantibodies to CTLA-4 could be used to augment the immune response totumors either alone or in combination with tumor antigen specificvaccines (Leach et al., Science, 271:1734 (1996)). Notably, transientautoimmune disease symptoms may be acceptable side-effects in patientshaving widely disseminated or inoperable cancers that are unresponsiveto conventional therapies.

[0076] Other contemplated tolerogens include viral antigens from virusesthat chronically infect humans including, but not limited to, HepatitisC virus (HCV), Hepatitis B virus (HBV), and HIV, chemokines, andmolecules associated with neoplasia and angiogenesis. By using theteachings described herein, one of skill in the art can present avariety of different tolerogens, including, nucleic acids, peptides,lipids, and carbohydrates, on biotinylated VLPs. For example, a sandwichapproach can be employed in which a biotinylated nucleic acid is firstbound to streptavidin and then the nucleic acid/streptavidin complex isbound to a biotinylated VLP. Similarly, using conventional chemistry,lipids can be joined to biotin, bound to streptavidin and bound tobiotinylated VLPs.

[0077] The ability to break B cell tolerance to small organic compoundswas established while performing experiments on biotinylated VLPs.Biotin is a vitamin and a self tolerogen in mice. We preparedbiotinylated VLPs, as described earlier, and injected these immunogensinto mice as before. The presence of anti-Biotin antibodies wasdetermined by an ELISA assay in which biotinylated BSA was used as thetarget antigen. As a negative control, the sera reactivity tounbiotinylated BSA was determined. The anti-biotin antibodies in thesera of three mice at titers of was 100, 100, and 10 and no reactivityto the unbiotinylated BSA was detected.

[0078] The compositions described above can be used as biotechnologicaltools, for example binding to an isolated cell of the immune system,which can provide a model system for the study of B cell tolerance butare preferably incorporated into therapeutics and prophylacticpharmaceuticals for the treatment and prevention of human disease. Thedisclosure below discusses several of the therapeutic and prophylacticembodiments of the invention.

[0079] Therapeutic and Prophylactic Applications

[0080] The compositions of the invention are suitable for treatment ofsubjects either as a preventive measure to avoid diseases such ascancer, viral infection or inflammatory conditions or as a therapeuticto treat subjects already afflicted with these maladies. Although anyonecould be treated with the agents of the invention as a prophylactic, themost suitable subjects are people at risk for diseases with mediatorsaccessible to Ab binding.

[0081] The pharmacologically active compounds of this invention can beprocessed in accordance with conventional methods of galenic pharmacy toproduce medicinal agents for administration to patients, e.g., mammalsincluding humans. They can be incorporated into a pharmaceutical productwith and without modification. Further, the manufacture ofpharmaceuticals or therapeutic agents that deliver the immunogens of theinvention by several routes are aspects of the invention.

[0082] The compounds of this invention can be employed in admixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application that do not deleteriously react with thecompositions of the invention. Suitable pharmaceutically acceptablecarriers include, but are not limited to, water, salt solutions,alcohols, gum arabic, vegetable oils, benzyl alcohols, polyetyleneglycols, gelatine, carbohydrates such as lactose, amylose or starch,magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil,fatty acid monoglycerides and diglycerides, pentaerythritol fatty acidesters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. Thepharmaceutical preparations can be sterilized and if desired mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,coloring, flavoring and/or aromatic substances and the like that do notdeleteriously react with the active compounds.

[0083] The effective dose and method of administration of a particularformulation can vary based on the individual patient and the stage ofthe disease, as well as other factors known to those of skill in theart. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, ED50/LD50.Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

[0084] The exact dosage is chosen by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors that may be taken into accountinclude the severity of the disease, age, and weight of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy.

[0085] Routes of administration include, but are not limited to,transdermal, parenteral, gastrointestinal, transbronchial, andtransalveolar. Parenteral routes of administration include, but are notlimited to, electrical or direct injection such as direct injection intoa central venous line, intravenous, intramuscular, intraperitoneal orsubcutaneous injection. Gastrointestinal routes of administrationinclude, but are not limited to, ingestion and rectal. Transbronchialand transalveolar routes of administration include, but are not limitedto, inhalation, either via the mouth or intranasally.

[0086] Compositions suitable for transdermal administration include, butare not limited to, pharmaceutically acceptable suspensions, oils,creams, and ointments applied directly to the skin or incorporated intoa protective carrier such as a transdermal device (“transdermal patch”).Examples of suitable creams, ointments, etc. can be found, for instance,in the Physician's Desk Reference. Examples of suitable transdermaldevices are described, for instance, in U.S. Pat. No. 4,818,540 issuedApr. 4, 1989 to Chinen, et al., herein incorporated by reference.

[0087] Compositions suitable for parenteral administration include, butare not limited to, pharmaceutically acceptable sterile isotonicsolutions. Such solutions include, but are not limited to, saline andphosphate buffered saline for injection into a central venous line,intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0088] Compositions suitable for transbronchial and transalveolaradministration include, but not limited to, various types of aerosolsfor inhalation. Devices suitable for transbronchial and transalveolaradministration are also embodiments. Such devices include, but are notlimited to, atomizers and vaporizers. Many forms of currently availableatomizers and vaporizers can be readily adapted to deliver thecompositions of the invention.

[0089] Compositions suitable for gastrointestinal administrationinclude, but not limited to, pharmaceutically acceptable powders, pillsor liquids for ingestion and suppositories for rectal administration.Due to the ease of use, gastrointestinal administration, particularlyoral, is the preferred embodiment of the present invention.

[0090] Several methods of treatment and prevention of human diseases areprovided, which involve administration of the pharmaceutical embodimentsof the invention. In these aspects, compositions of the invention areincorporated into pharmaceuticals and are administered to patients inneed. By one approach, a subject at risk for contracting HIV infectionor another chronic viral infection or a subject already infected withHIV or another chronic viral infection is identified by conventionaldiagnostic assays and then a therapeutically or prophylacticallybeneficial amount of a pharmaceutical of the invention is administeredto the subject. A similar approach can be employed to treat and/orprevent chronic inflammatory disease. That is identifying a subject inneed and then administering a pharmaceutical comprising a composition ofthe invention. Other methods of the invention include an approach toraise high titer neutralizing antibodies. Accordingly, agents (e.g., acomposition of the invention) are identified for their ability to breakB cell tolerance and are subsequently administered to a subject in need.Additional embodiments include a method to make monoclonal andpolyclonal antibodies to a composition of the invention. These novelantibodies can also be incorporated into pharmaceuticals andadministered to patients in need for the treatment and prevention ofhuman disease. The disclosure below provides more discussion of theseapproaches.

[0091] Preparation of Antibodies to Chimeric and Conjugated VLPs

[0092] Following construction of a chimeric or conjugated VLP, thesecompositions can be used to generate antibodies. (See Example 10).Antibodies that recognize a chimeric or conjugated VLP have many usesincluding, but not limited to, biotechnological applications,therapeutic/prophylactic applications, and diagnostic applications. Suchantibodies include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fab fragments and fragments produced by a Fabexpression library. Neutralizing antibodies, i.e., those that inhibitCCR5-mediated adhesion, are especially preferred for therapeutics.

[0093] For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc can be immunized by injection with a chimericor conjugated VLP. Depending on the host species, various adjuvants canbe used to increase immunological response. Such adjuvants include butare not limited to Freund's, mineral gels such as aluminum hydroxide,and surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvumare potentially useful adjuvants. However, VLP-based immunogens can alsoincrease the titer of antibodies to tolerogens without the addition ofadjuvants.

[0094] Monoclonal antibodies to a chimeric or conjugated VLP can beprepared using any technique that provides for the production ofantibody molecules by continuous cell lines in culture. These includebut are not limited to the hybridoma technique originally described byKoehler and Milstein (Nature, 256:495-497 (1975), the human B-cellhybridoma technique (Kosbor et al., Immunol Today, 4:72 (1983); Cote etal., Proc Natl Acad Sci, 80:2026-2030 (1983), and the EBV-hybridomatechnique Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R.Liss Inc, New York N.Y., pp 77-96 (1985), all articles hereinincorporated by reference. In addition, techniques developed for theproduction of “chimeric antibodies”, the splicing of mouse antibodygenes to human antibody genes to obtain a molecule with appropriateantigen specificity and biological activity can be used. (Morrison etal., Proc Natl Acad Sci, 81:6851-6855 (1984); Neuberger et al., Nature,312:604-608 (1984); and Takeda et al., Nature, 314:452-454 (1985), allarticles herein incorporated by reference. Alternatively, techniquesdescribed for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies directed toa chimeric or conjugated VLP, herein incorporated by reference.Antibodies can also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al., Proc Natl Acad Sci, 86: 3833-3837 (1989), and Winter G.and Milstein C, Nature, 349:293-299 (1991), all articles hereinincorporated by reference.

[0095] Antibody fragments that contain specific binding sites for achimeric or conjugated VLP can also be generated. For example, suchfragments include, but are not limited to, the F(ab′)₂ fragments thatcan be produced by pepsin digestion of the antibody molecule and the Fabfragments that can be generated by reducing the disulfide bridges of theF(ab′)₂ fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity. (Huse W. D. et al., Science,256:1275-1281 (1989), herein incorporated by reference).

[0096] By one approach, monoclonal antibodies to a chimeric orconjugated VLP are made as follows. Briefly, a mouse is repetitivelyinoculated with a few micrograms of the selected protein or peptidesderived therefrom over a period of a few weeks. The mouse is thensacrificed, and the antibody producing cells of the spleen isolated. Thespleen cells are fused in the presence of polyethylene glycol with mousemyeloma cells, and the excess unfused cells destroyed by growth of thesystem on selective media comprising aminopterin (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution placedin wells of a microtiter plate where growth of the culture is continued.Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as ELISA,as originally described by Engvall, E., Meth. Enzymol., 70:419 (1980),herein incorporated by reference, and derivative methods thereofSelected positive clones can be expanded and their monoclonal antibodyproduct harvested for use. Detailed procedures for monoclonal antibodyproduction are described in Davis, L. et al. Basic Methods in MolecularBiology Elsevier, New York. Section 21-2.

[0097] Polyclonal antiserum containing antibodies to heterogenousepitopes of a single protein can be prepared by immunizing suitableanimals with the expressed protein or peptides derived therefromdescribed above, which can be unmodified or modified to enhanceimmunogenicity. Effective polyclonal antibody production is affected bymany factors related both to the antigen and the host species. Also,host animals vary in response to site of inoculations and dose, withboth inadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng level) of antigen administered at multipleintradermal sites appears to be most reliable. An effective immunizationprotocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin.Endocrinol. Metab., 33:988-991 (1971).

[0098] Booster injections can be given at regular intervals, andantiserum harvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al., Chap. 19 in: Handbook of ExperimentalImmunology D. Wier (ed) Blackwell (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibodypreparations prepared according to either protocol are useful inquantitative immunoassays that determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively. Additionally, a chimeric orconjugated VLP can be used to induce antibody production in humans, asdiscussed throughout this disclosure. Accordingly, a chimeric orconjugated VLP can be joined to or administered with another protein,carrier, support, or adjuvant so as to generate a pharmaceutical orvaccine that will induce potent immune response.

[0099] The following Example describes the procedures that were used toprepare and express a polynucleotide encoding a chimeric L1-CCR5 proteinthat self assembled into capsomeric structures. The below-describedprocedure involved modifying an L1 encoding polynucleotide toincorporate an amino acid sequence encoding a CCR5 peptide fragment. Inthis exemplary demonstration, sixteen codons from the C57B1/6 (B6) mouseCCR5 (mCCR5) first extracellular loop were separately inserted into oneof three regions of the BPV-1 L1 sequence corresponding to the sites ofvirus-neutralizing epitopes. The positions of these epitopes previouslyhad been deduced by alignment of polypeptide sequences of various humanpapillomaviruses. The three non-contiguous regions of L1 that receivedthe CCR5 sequence have been described by Ludmerer et al., J. Virol.,70:4791 (1996); by Ludmerer, et al., J. Virol., 71:3834 (1997); and byRoden, et al., J. Virol., 71:6247 (1997). Since the amino acids at thesesites were likely to be expressed on the capsid surface, analogous sitesin BPV-1 L1 were targeted for peptide insertion. This ensured that theportion of the chimeric L1 protein that included the CCR5 peptidesequence would be surface-expressed and available for efficientpresentation to the humoral immune system.

[0100] Example 1 describes the method used to create a chimeric L1-CCR5protein that self assembled into antigenic particles.

EXAMPLE 1 Construction of a Chimeric Protein Capable of Self Assemblinginto Antigenic Particles

[0101] Polynucleotides encoding three different L1-CCR5 chimeras(designated “L1-CCR5 chimera 1”, “L1-CCR5 chimera 2” and “L1-CCR5chimera 3”) were prepared by overlap extension PCR mutagenesisessentially according to the technique described by Ho et al., in Gene,77:51 (1989). A polynucleotide encoding BPV-1 L1 (Chen et al., Nature,299:557 (1982)) was cloned as an EcoRI/KpnI fragment into complementarysites of the multiple cloning site of the baculovirus pFastBac1expression vector (Gibco BRL, Gaithersberg, Md.). Portions of the BPV-1L1 sequence in each of the three chimeras were replaced by a sequenceencoding the first extracellular loop of C57B1/6 mCCR5. The polypeptidesequence from the mCCR5 protein had the sequence:His-Tyr-Ala-Ala-Asn-Glu-Trp-Val-Phe-Gly-Asn-Ile-Met-Cys-Lys-Val (SEQ IDNO:1) (Boring et al., J. Biol Chem., 271:7551 (1996)). In L1-CCR5chimera 1, the sequence encoding L1 amino acids 130-136 was replaced bythe mCCR5 sequence. In L1-CCR5 chimera 2, the sequence encoding L1 aminoacids 275-285 was replaced by the mCCR5 sequence. In L1-CCR5 chimera 3,the sequence encoding L1 amino acids 344-350 was replaced by the mCCR5sequence. The final clones were verified by restriction digest analysisand by nucleotide sequence analysis of the PCR-amplified region.

[0102] Recombinant baculovirus stocks containing the genes coding forthe chimeric L1-CCR5 proteins or wild type BPV-1 L1 were generated usingthe GIBCO BRL baculovirus system, as described by the manufacturer.Papillomavirus-like particles were purified from recombinantbaculovirus-infected Sf9 cells as described previously. (Kirnbauer, R.et al., Proc. Natl. Acad. Sci. USA, 89:12180-12184 (1992); Greenstone,H. L. et al., Proc. Natl. Acad. Sci. USA, 95:1800-1805 (1998)). Thegeneral morphology of the particle preparations was analyzed by mobilityassay using an FPLC Superose 6 gel filtration column (Pharmacia Biotech,Uppsala, Sweden). Eluate was collected in one ml fractions. The voidvolume of this column is 8 ml. Previously, it was determined that wildtype L1 VLPs predominantly elute in fraction 9 of the column, L1capsomeres elute in fraction 15, and L1 monomers elute in fractions19-21 (Okun, M. M. et al., submitted for publication). Column fractionswere assayed for the presence of L1 by Western blot.

[0103] Example 2 describes the methods used to confirm that a chimericL1-CCR5 protein self-assembled into capsomeric structures.Interestingly, the L1-CCR5 particles described below were shown byelectron microscopy to be somewhat smaller than VLPs formed of wild typeL1 proteins.

EXAMPLE 2 Preparation of Chimeric Capsomeric Structures

[0104] The three above-described L1-CCR5 chimeras were isolated by FPLCSUPEROSE 6 gel filtration column chromatography (Pharmacia Biotech,Uppsala, Sweden). Column fractions of 1 ml each were assayed for thepresence of L1 by Western blotting using a 10% polyacrylamide gel underdenaturing conditions. Control procedures indicated that wild type L1VLPs predominantly eluted in the column fraction 9, that L1 capsomereseluted in fraction 15, and that L1 monomers eluted in fractions 19-21.L1-CCR5 protein from preparations of chimeras 2 and 3 was detectedpredominantly in fraction 15. These results suggested that L1-CCR5chimera 2 and L1-CCR5 chimera 3 proteins failed to assemble into higherorder structures. Based on these results, we selected the L1-CCR5chimera 1 for subsequent procedures. Purified particles were examinedusing electron microscopy by first adsorbing the particles tocarbon-coated grids, staining with 1% uranyl acetate and then examiningthe grids using a Philips electron microscope model EM 400RT at 36,000×magnification.

[0105] Results of these procedures indicated that the L1-CCR5 chimera 1protein eluted in a column fraction known to contain assembledparticulate structures. Examination of chimera 1 particles by electronmicroscopy revealed particles having diameters of approximately 28 nmwhile wild type L1 VLPs had diameters of approximately 55 nm. The 28 nmdiameter suggests that the particles were composed of 12 capsomereswhile the larger diameter structures formed of wild type L1 proteinswere composed of 72 capsomeres. Morphologically, the L1-CCR5 chimera 1particles resembled polyomavirus 12 ICOSA shells (T=1 particles) whichare composed of a regular array of 12 pentameric capsomeres of thepolyomavirus major coat protein VP1 and can be generated upon in vitroreassembly of VP1 capsomeres at high ionic strength (Salunke, et al.,Biophys. J., 56:887 (1989)). Small particles of a size similar to theL1-CCR5 particles were found as minor components of wild type BPV-1 L1VLP preparations. Although the L1-CCR5 particles were smaller than wildtype VLPs, they possessed at least some characteristics of wild typeVLPs that wild type capsomeres lack. In particular, L1-CCR5 particleshemagglutinated mouse red blood cells and displayed ELISA reactivity toa BPV-1 neutralizing monoclonal antibody (#9), which specifically boundto particles but not capsomeres (Roden, et al., J. Virol., 68:7570(1994)).

[0106] The following Example illustrates how peptide sequences displayedas ordered arrays on capsomeric structures can stimulate humoral immuneresponses, even against central antigens. As indicated below, miceadministered with L1-CCR5 particles responded by producing mCCR5specific antibodies. Significantly, the results indicated that theimmunization overcame B cell tolerance to the peptide without affectingtolerance to endogenous cellular CCR5. The anti-self antibodies inducedby the immunogenic particles bound native mCCR5, blocked binding of aCCR5 ligand and inhibited HIV-1 infection through a chimeric CCR5protein that contained the mCCR5 peptide.

[0107] Example 3 describes the methods used to demonstrate that chimericL1-CCR5 particles could be used as immunogens to induce anti-CCR5antibodies.

EXAMPLE 3 Stimulation of a Tolerance Breaking Immune Response

[0108] To prepare antisera, C57B1/6 mice were administered in animmunization protocol with either: L1-CCR5 particles, wild type BPV-1 L1VLPs, or a synthetic CCR5 peptide representing the first extracellularloop of mCCR5 that was coupled to keyhole limpet hemocyanin (KLH) usingan IMJECT activated immunogen conjugation kit (Pierce, Rockford, Ill.).In some cases, mice were administered with L1-CCR5 particles that hadbeen denatured by boiling for 2 minutes in the presence of 1% SDS. Micewere innoculated intradermally with 10 μg of antigen three times attwo-week intervals. In most cases, serum samples were collected twoweeks after the final boost. When adjuvant was used, the antigen wasprepared in Freund's complete adjuvant for the initial injection, and inFreund's incomplete adjuvant for subsequent injections. Serum sampleswere tested for reactivity against the CCR5 peptide and against wildtype VLPs using a quantitative ELISA protocol to detect IgG antibodyagainst BPV-1 VLPs. The ELISA was performed using the proceduredescribed by Kirnbauer et al., in J. Natl. Cancer Inst., 86:494 (1994).A synthetic peptide representing the first extracellular loop of mCCR5was prepared and coupled to bovine serum albumin (BSA) as a carrierprotein. Anti-CCR5 specific IgG was detected by binding 300 ngBSA-coupled CCR5 peptide in 50 μl phosphate buffered saline (PBS) toeach well of a 96-well IMMULON II microtiter plate (Dynatech; Chantilly,Va.) for 2 hours at 37 EC. After washing three times with PBS, the wellswere blocked for 2 hours with 50 μl of PBS containing 0.5% nonfat drymilk plus 1% newborn calf serum at room temperature. After blocking, thewells were again washed three times with PBS thereafter. Mouse serum wasserially diluted in PBS plus 0.5% nonfat dry milk. Diluted serum samples(50 μl) were applied to the wells after removing the final PBS wash.Plates were incubated at room temperature for 2.5 hours with gentlerocking. After five washes, 50 μl of horseradish peroxidase conjugatedgoat anti-mouse IgG (Boehringer Mannheim; Indianapolis, Ind.) diluted1:10,000 in 0.5% milk-PBS was added to the wells. Plates were incubatedat room temperature for 1 hour with gentle rocking and then washed threetimes. The ABTS peroxidase substrate (50 μl) (Boehringer Mannheim) wasadded to the plate, incubated for 45 minutes at room temperature, andoptical densities (ODs) read at 405 nm using a THERMO MAX microplatereader. OD₄₀₅ values greater than twice background (usually greater than0.1) were considered positive.

[0109] The results presented in FIG. 1A indicated that serum samplesfrom mice administered with L1-CCR5 particles had high anti-CCR5 ELISAtiters. In contrast, control mice administered with wild type VLPs hadno ELISA reactivity, as expected. Anti-CCR5 titers ranged from 3×10³ to3×10⁴ in the three animals that had been administered with the immunogenin combination with Freund's adjuvant, and measured 3×10³ in the twoanimals that had received the immunogen without adjuvant. Miceadministered with denatured L1-CCR5 particles in combination withadjuvant showed no evidence for mCCR5 peptide-specific antibodies. Thelack of reactivity of the denatured L1-CCR5 particles was limited to theCCR5 peptide since, as indicated by the results presented in FIG. 1B,the denatured material elicited high titers of anti-L1 antibodies. Thesefindings clearly demonstrated that adult mammals retained the ability toproduce antibodies specific for central self antigens.

[0110] While the foregoing results indicated that the L1-CCR5 particleselicited antibodies specific for the CCR5 peptide, further testing wasconducted to verify that the antibodies also recognized cell-associatedmCCR5 protein when displayed in its native conformation. This wasaccomplished using flow cytometric analysis (FACS) to show that theanti-CCR5 antibodies bound mCCR5 expressed on the cell surface.

[0111] Example 4 describes the methods used to demonstrate thatantibodies raised against the L1-CCR5 particles bound authentic mCCR5receptor protein expressed on cell surfaces.

EXAMPLE 4 Binding of Native Antigen by Autoantibodies Stimulated inResponse to a Self Antigen Incorporated into a Capsomeric Structure

[0112] Total IgG from pooled mouse sera was affinity purified over aProtein G column (Pierce) using procedures that will be familiar tothose having ordinary skill in the art. Column fractions containing IgGwere pooled and then concentrated using a CENTRICON-30 spin column(Amicon; Beverly, Mass.). Antibody binding assays were conducted usingtransfected human cells that expressed a recombinant mouse CCR5receptor. The binding of antibodies raised against L1-CCR5 particlescould not be tested easily using primary cultures of mouse cells becausethese cells expressed high levels of Fcy receptors and yielded highbackground levels of binding due to interactions with non-specific mouseIgG. Accordingly, for flow cytometry analysis a mouse CCR5 expressionvector was transiently expressed in HeLa-MAGI cells by transfectionusing a LIPOFECTAMINE PLUS transfection kit (Gibco BRL; Gaithersberg,Md.). pcDNA3 derived plasmids containing mCCR5 cloned from B6 mice and ahuman-mouse CCR5 chimera containing the first extracellular loop ofmCCR5 in a background of human CCR5 were prepared as described byKuhmann et al., in J. Virol., 71:8642 (1997). Monolayers were detachedby gentle scraping in the presence of 5 mM EDTA at 48 hourspost-transfection. Cells were washed three times in staining buffer (PBSplus 0.5% BSA). Approximately 10⁵ cells were resuspended in 25 μlstaining buffer plus 1 μg of mouse IgG and then incubated 45 minutes at4 EC. Cells were washed three times with staining buffer, resuspended in25 μl of staining buffer plus 250 ng fluorescein (FITC)-labelled goatanti-mouse IgG (Jackson Immunoresearch; West Grove, Pa.), and incubatedfor 30 minutes at 4 EC. Cells were washed three times with stainingbuffer and finally resuspended in 0.5 ml staining buffer in preparationfor FACS analysis. As a control, cells were stained with 500 ngFITC-labelled mouse anti-human CCR5 monoclonal antibody (mAB182) (R & DSystems; Minneapolis, Minn.) according to the manufacturer'sspecifications. FACS analysis was performed using a FACSCALIBUR andCELLQUEST software (Becton Dickinson; San Jose, Calif.). Specificbinding was measured relative to staining of control cells transfectedwith the pcDNA3 vector.

[0113] Results from these procedures indicated that autoantibodiesstimulated in response to administration of L1-CCR5 capsomeric particlesspecifically bound recombinant mCCR5 receptors expressed on the surfaceof transfected HeLa-MAGI cells. FIG. 2A shows that IgG from miceimmunized with L1-CCR5 bound specifically with high affinity totransfected cells that expressed the mCCR5 receptor but not to cellstransfected with vector alone. Cells expressing mCCR5 did notsubstantially bind antibodies that were stimulated in response toimmunization with virus-like particles formed by wild type L1 (FIG. 2B)or a monoclonal antibody (mAB 182) specific for the second extracellularloop of human CCR5 (FIG. 2C), as expected. As a control for antibodyspecificity, mice were administered with mCCR5 peptide that had beencoupled to keyhole limpet hemocyanin (KLH). While these mice respondedby producing anti-CCR5 peptide antibodies having ELISA titers of 10⁵against a BSA-coupled, purified IgG failed to bind cells expressingmCCR5 (FIG. 2D). In aggregate, these results indicated that antibodiesraised in response to immunization with L1-CCR5 capsomeric particlesfunctioned as true auto-antibodies because they specifically bound cellsurface-expressed native mCCR5, in contrast to the antibodies raisedagainst the KLH-CCR5 peptide.

[0114] The ability of antibodies raised against L1-CCR5 particles tobind native mCCR5 was further examined by testing for competition withthe ¹²⁵I-labelled human RANTES chemokine ligand for binding totransfected HeLa-MAGI cells expressing mCCR5. The mouse chemokinesMIP-1α, MIP-1β and RANTES are ligands for mCCR5. In addition, the humanhomologs of MIP-1β and RANTES are able to bind mCCR5 (Meyer et al., J.Biol. Chem., 271:14445 (1996); Nibbs et al., J. Biol. Chem., 272:12495(1997)). As described in the following Example, cells were incubatedwith 0.5 nM iodinated RANTES in the absence or presence of dilutions ofmouse sera three days after transfection with a mCCR5 expressionconstruct.

[0115] Example 5 describes the methods used to demonstrate thatautoantibodies raised against the CCR5 receptor inhibited ligand bindingto the receptor.

EXAMPLE 5 Autoantibodies Specific for a Receptor Inhibit Ligand Binding

[0116] HeLa-MAGI cells were transiently transfected with the mCCR5expression plasmid using a CaPO₄ transfection kit that was purchasedfrom Stratagene Cloning Systems (La Jolla, Calif.). At two dayspost-transfection 10⁵ cells were transferred into individual wells of a24-well tissue culture plate. The following day cells were washed twicein cold PBS and then resuspended in 150 μl cold binding buffer (25 mMHEPES (pH 7.2), 5 mM MgCl₂, 1 mM CaCl₂, 0.5% (wt/vol) BSA). Cells wereincubated for 4 hours at 4 EC with 0.5 nM ¹²⁵I-labelled human RANTES(Amersham; Arlington Heights, Ill.) in the absence or presence ofvarious dilutions of mouse sera. To remove small molecules, mouse serawas buffer exchanged into binding buffer using MICRO BIO-SPINCHROMATOGRAPHY-6 columns (Bio-Rad, Hercules, Calif.) prior to conductingthe binding assays. As a control, some binding assays were performed inthe presence of 50 nM or 500 nM non-iodinated human RANTES (R & DSystems). Reactions were stopped by washing the wells four times withcold binding buffer plus 0.5 M NaCl. Cells were lysed by adding 0.5 ml1% SDS, and the lysates transferred to counting vials. Boundradioactivity was counted for 1 minute in a Beckman Gamma 5500B counter.

[0117] Results from these procedures confirmed that autoantibodiesraised against the L1-CCR5 particles specifically bound cell surfaceexpressed CCR5 and inhibited ligand binding to the receptor. Moreparticularly, the graphic results shown in FIG. 4 indicated that a 1:30dilution of L1-CCR5 sera displaced approximately 66% of the iodinatedhuman RANTES (similar to the displacement observed using a 100-foldexcess of cold RANTES), compared with 37% displacement with a 1:30dilution of wild type L1 VLP sera. The 1:75 and 1:150 dilutions ofL1-CCR5 sera displaced 25% and 17% of the iodinated RANTES,respectively, whereas no significant displacement was observed usingcontrol sera at these dilutions. Maximally bound iodinated RANTES wasdetermined by assaying for binding in the absence of sera, andcorresponded to approximately 2550 cpm indicated by the dashedhorizontal line in FIG. 3. Non-specific binding of iodinated RANTES(approximately 1300 cpm) was determined by assaying for binding in a1000-fold excess (500 nM) of cold (non-iodinated) human RANTES. Datashown in FIG. 4 represents the average of duplicate wells from oneexperiment. Previous studies have suggested MIP-1α, MIP-1β and RANTESbind to the second extracellular loop of human (h) CCR5, since theirbinding was blocked by monoclonal antibody to this portion of themolecule but not by an antibody specific for the amino terminus of hCCR5(Wu et al., J. Exp. Med., 186:1373 (1997)). The findings presentedherein indicated that antibodies having binding specificity for thefirst extracellular loop of mCCR5, which is located between these twosites, advantageously inhibited RANTES binding and further provided away to stimulate formation of these autoantibodies in vivo.

[0118] To further investigate the utility of the above-describedautoantibodies, we investigated whether the inhibition of ligand bindingobserved in the foregoing Example correlated with inhibition of viralinfection of target cells. Monoclonal antibody studies have implicatedthe second extracellular loop and the amino terminal region of hCCR5,and studies of chimeric receptors have indicated that the first andthird extracellular loops of CCR5 also contribute to receptorinteraction with HIV-1 (Wu et al., J. Exp. Med., 186:1373 (1997); Ruckeret al., Cell, 87:437 (1996); Atchison et al., Science, 274:1924 (1996);Alkhatib et al., J. Biol. Chem., 272:19771 (1997); Picard et al., J.Virol., 71:5003 (1997); Ross et al., J. Virol., 72:1918 (1998)).Although mCCR5 does not function as an HIV-1 co-receptor, a human-mousechimeric receptor (HMHH), which contains the first extracellular loop ofmCCR5 (the B6 mouse sequence) in a background of hCCR5, has co-receptoractivity when expressed in human cell lines (Kuhmann et al., J. Virol.,71:8642 (1997)). Accordingly, this chimeric receptor was used in thefollowing Example to test whether anti-L1-CCR5 sera could block M-tropicHIV-1 infection.

[0119] The results presented in the following Example have strongbearing on the inhibition of HIV infection because even partialreduction in CCR5 expression can have clinically significant effects.This is true because HIV-1 infected individuals who are heterozygous foran inactive CCR5 allele exhibit delayed progression to AIDS (Liu et al.,Cell, 86:367 (1996); Samson et al., Nature, 382:722 (1996); Winkler etal., Science, 279:389 (1998)).

[0120] Example 6 describes the methods used to demonstrate thatautoantibodies raised in response to L1-CCR5 particles inhibitedinfection of target cells by M-tropic HIV-1. Since a chimericmouse-human CCR5 receptor was used in these procedures, the initial stepinvolved confirming that the above-described anti-CCR5 autoantibodiesrecognized the chimeric receptor.

EXAMPLE 6 Anti-Receptor Autoantibodies Inhibit HIV Infection of TargetCells

[0121] To confirm that IgG purified from L1-CCR5 sera bound thehuman-mouse chimeric receptor, FACS analysis was performed on HeLa-MAGIcells transiently transfected with the HMHH expression constructdescribed by Kuhmann et al., in J. Virol., 71:8642 (1997). Theexpression construct was transfected into recipient cells 2 days priorto staining with either L1-CCR5 IgG, wild type L1 VLP IgG or a positivecontrol anti-human CCR5 monoclonal antibody. The results presented inFIGS. 2E-2G indicated that positive binding was obtained using serum IgGfrom mice administered with L1-CCR5 particles as well as with a positivecontrol monoclonal antibody specific for the second extracellular loopof human CCR5. However, IgG from mice administered with wild type L1 VLPdid not bind HMHH, as expected.

[0122] Based on the foregoing results, sera from L1-CCR5 mice weretested for the ability to inhibit infection of the M-tropic BaL strainof HIV-1, using a single replication cycle assay and the HeLa-MAGIindicator cell line. HeLa-MAGI cells, described by Kimpton et al., in J.Virol., 66:2232 (1992), were transiently transfected with the chimerichuman-mouse CCR5 expression vector using a commercially obtained CaPO₄transfection kit (Stratagene Cloning Systems). Two days aftertransfection, and the day prior to infection, the indicator cells wereseeded to 24-well plates at 6.5×10⁴ cells per well in complete DMEM.Some infections were performed in the presence of pooled mouse serawhich had been buffer-exchanged into PBS using MICRO BIO-SPINCHROMATOGRAPHY-6 columns (Bio-Rad). Prior to infection, cells wereincubated in a total volume of 140 μl in complete DMEM with 10 μg/mlDEAE-dextran plus dilutions of sera for 30 minutes at 4 EC. Afterincubation, virus was added to each well to give a total volume of 150μl. Cells were incubated for 2 hours at 37 EC, then 1 ml of completeDMEM was added to each well. At 3 days post-infection cells were stainedwith X-gal and an infectious dose determined by counting the number ofblue nuclei in infected wells. Inhibition of viral entry was scored bycomparing the average number of blue nuclei in the presence of sera withaverage number of infectious centers in the absence of sera. Typically,enough infectious virions to lead to 50-75 infectious blue centers incontrol (no sera) wells were used in each infection. All assays wereperformed in duplicate. Over passage, the efficiency of infection oftransfected HeLa-MAGI cells markedly decreased, presumably because ofreduced CD4 expression. Therefore, all infections were performed onrecently thawed HeLa-MAGI cells.

[0123] Results of these procedures, presented graphically in FIG. 4,showed that serum antibodies raised against the self CCR5 antigen, andthat were shown to recognize native antigen and that inhibitedligand-receptor interactions, also inhibited viral infection of targetcells. Indicator cells that were transiently transfected with the HMHHexpression construct and contacted with HIV-1 BaL in the presence ofL1-CCR5 sera dilutions of 1:15, 1:30, and 1:75 exhibited 65%, 50%, and45% neutralization of infectivity. At the same dilutions, control serafrom wild type L1 VLP mice exhibited some non-specific neutralization,but only at a level of 25% at the 1:15 dilution and 15% at 1:30 and1:75. Indicator cells infected with HIV-1 BaL in the presence of 50μg/ml of a monoclonal antibody specific for human CCR5 (mAB182) that wasused as a positive control exhibited approximately 50% neutralization.Thus, the anti-CCR5 autoantibodies produced in accordance with theprocedure set forth above effectively inhibited infection of susceptiblecells by HIV-1.

[0124] We have shown how to break B cell tolerance to a self antigen bypresenting it in a context that mimics the ordered surface antigens ofan infectious virus. To do this, we substituted a dominantvirus-neutralizing epitope on the surface of papillomavirus L1 VLPs witha peptide sequence from a self protein. More specifically, we engineeredinto a putative neutralizing epitope in the L1 of bovine papillomavirustype 1 (BPV-1) (Ludmerer et al., J. Virol., 70:4791 (1996)) a peptidesequence 16 amino acids in length which corresponded to the firstexternal loop of the mouse chemokine receptor CCR5. This chimeric L1assembled into particles having ordered arrays of capsomeres that couldbe used as immunogens for stimulating humoral immune responses againstthe chimeric L1-CCR5 protein.

[0125] Mice immunized with VLPs composed of chimeric L1-CCR5 proteinsubunits were maintained to determine the long term effects of theimmunization, including any pathological consequences of autoantibodyproduction. At six months post immunization, the immunized mice weighedthe same as control animals and appeared outwardly healthy. An autopsyof the mouse with the highest anti-CCR5 titers did not reveal anyindications of autoimmune disease. The CCR5 antibody titers in thevaccinated mice were initially stable but then declined slowly, inparallel with the responses to L1. These results suggest that thecellular CCR5 neither activates nor tolerizes the chimeric VLP induced Bcell response to the CCR5 peptide.

[0126] The following Example describes how autoantibodies directed to acentral self antigen can be stimulated in a mammal other than a mouse.In the exemplary case illustrated below a composition and method forinducing production of anti-macaque CCR5 antibodies is described.

EXAMPLE 7 Stimulation of an Autoimmune Response in Macaques

[0127] A recombinant expression construct encoding a chimeric L1-CCR5protein which includes a portion of the macaque CCR5 polypeptidesequence was first prepared essentially according to the procedure setforth under Example 1. The resulting expression construct was introducedinto recipient Sf9 cells where protein encoded by the recombinant vectorwas produced. Capsomeric structures representing self-assembledaggregates of the chimeric L1-CCR5 protein produced in the recipientcells were purified by sucrose gradient and CsCl gradientcentrifugation. In a parallel procedure, wild type VLPs composed of wildtype L1 protein also were prepared and purified for use as a controlimmunogen. The control immunogen does not contain the macaque CCR5polypeptide sequence that is present in the L1-CCR5 chimera. Purifiedwild type VLPs or chimeric L1-CCR5, combined with adjuvant capsomericstructures, give control and test immunogenic compositions,respectively. These compositions are separately injected intradermallyinto macaques according to a standard immunization protocol such as thatdescribed under Example 3. In one instance the animals are administeredwith the immunogenic compositions three times at two week intervals.Serum samples taken from the two animals periodically from a time beforethe initial immunization indicated no evidence for CCR5-bindingantibodies before immunization. Serum samples from the control animalshow no evidence for CCR5-binding antibodies even several weeks afterthe final administration of the wild type L1 VLP immunogeniccomposition. In contrast, serum samples from the animal administeredwith capsomeric structures that included the L1-CCR5 chimera containsignificant levels of anti-CCR5 antibodies. (FIG. 5). These resultsconfirmed that the L1-CCR5 capsomeric structures have the desiredimmunogenicity and that the effect is antigen-specific.

[0128] We have previously shown that addition of other papillomaviruspolypeptides to the VLPs, as fusions of the L2 minor capsid protein, caninduce a strong cell mediated immune response against these viralpeptides and the production of specific antibodies against the insertedpeptide (Greenstone et al., Proc Natl Acad Sci USA, 95:1800 (1998); H.L. Greenstone Ph.D. Thesis (1998), The Johns Hopkins University,Baltimore, Md.). Both induction of high titer antibody responses and MHCI restricted CTL responses can be induced by low dose inoculation ofVLPs in the absence of adjuvant. In view of the findings presentedabove, the capacity of VLPs to induce potent immune responses againstviral epitopes is probably related to their ability to interact withcell surfaces and to present epitopes as an ordered array of repetitivestructure.

[0129] We have had success generating L2 chimeras of viral proteins.Significantly, all of the fusions were compatible with co-assembly intofull-sized L1 VLPs that could be efficiently recovered as particles(Greenstone et al., Proc Natl Acad Sci USA, 95:1800 (1998)). Largeinserts, even representing 42 kDa full-length proteins, in the L2protein were compatible with VLP assembly. The ability of the L2 toaccept inserts of this size is attributed to the fact that L2 does notcontribute to the structural integrity of the VLP and so can toleratesubstantial modification without compromising particle self-assembly.While it is likely that L2 has an ordered structure in the VLPs, itsspacing probably is not as close as the spacing of L1. Although thelocation of L2 in the papillomavirus capsid has not been definitivelydetermined, we have experimental evidence showing that L2 is located atthe twelve vertices of the icosahedral capsid. This would place L2 andany peptide inserted therein at a repeat distance of approximately 300angstroms.

[0130] Since the papillomavirus L2 protein can accommodate large insertsof extraneous polypeptide sequence and still incorporate into virus-likeparticles, L1/L2 chimeras having full-length self proteins inserted atthe site of L2 can be prepared and used as immunogens. In a preferredembodiment the target polypeptide is fused to the first 110 amino acidsof the BPV L2 protein. This presents the insert sequence on the capsidexterior when assembled into L1 VLPs. Indeed, this approach can be usedto prepare chimeric VLPs that can be used as immunogens for stimulatingproduction of autoantibodies against TNF-α.

[0131] The following two Examples describe how to make mouse TNF-α VLPsboth as L2 chimeras and as streptavidin fusions. Based upon the knownatomic structure of the protein (Eck et al., J. Biol. Chem., 264:17595(1989)), L1 chimeras also can be prepared by inserting TNF-α peptidesthat include epitopes to which functionally neutralizing antibodiesbind. Serum from TNF-α-VLP vaccinated mice can be tested for reactivityagainst mouse TNF-α in an ELISA assay and for inhibition of TNF-αinduced cytolysis of L929 cells in vitro (Takasaki et al., NatureBiotech, 15:1266 (1997)). The VLPs displaying the TNF-α polypeptidesequence can also be used to vaccinate DBA/1 mice having collagen typeII RA. The effect of this treatment on the course of disease can bemonitored using standard paw swelling and histological analyses(Thorbecke et al., Proc. Natl. Acad. Sci. USA, 89:7375 (1992)).

[0132] Example 8 describes a method that can be used to prepare chimericL1/L2 particles for use as immunogens.

EXAMPLE 8 Chimeric L1/L2 Particles to Stimulate Production ofAutoantibodies

[0133] Using standard techniques that will be familiar to those havingordinary skill in the art of molecular cloning, genetic constructsencoding L2-mouse TNF-α polypeptide chimeras are prepared and expressedwithin transfected cells as chimeric proteins. The chimeric proteins arethen co-assembled into L1 virus-like particles to result in chimericL1/L2 VLPs. The L1/L2 VLPs are purified, combined with an adjuvant andadministered to test animals in an immunization protocol. As controls,the L2-TNF-α chimeric protein and soluble TNF-α are injected alone.Serum samples from the mice administered with the chimeric L1/L2 VLPpreparation contain anti-TNF-α antibodies that are detectable in anELISA assay. In contrast, serum samples from control animals do notcontain anti-TNF-α antibodies. This result indicates that chimeric L1/L2particles that include chimeric L2 proteins are useful for stimulatingproduction of autoantibodies.

[0134] Example 9 describes a method for making and using immunogenicvirus-like particles wherein a self polypeptide sequence is joined tothe proteins which make up the particle through a biotin linkage. Inthis instance, pre-formed VLPs are biotinylated and then contacted withan streptavidin-linked self peptide. This manipulation is possiblebecause wild type L1 VLPs are quite stable once they are formed.

EXAMPLE 9 Virus-Like Particles Incorporating Self Polypeptides Through aBiotin Linkage

[0135] Purified wild type L1 VLPs were biotinylated usingsulfo-NHS-Biotin reagents according to the manufacturer's instructions(Pierce). Biotinylated VLPs were purified from free biotin by separationon a 24%-54% linear sucrose gradient. Preliminary experimentsdemonstrated that L1 VLPs were heavily biotinylated. This observationindicated that there was at least one exposed lysine for biotinattachment on each L1. A mouse TNF-α polypeptide was conjugated to theVLPs as a streptavidin fusion protein. Streptavidin was generally usefulfor efficiently attaching any polypeptide of choice to the biotinylatedVLPs. The polypeptide sequence from the mouse TNF-V protein had thesequence:Ser-Ser-Gln-Asn-Ser-Ser-Asp-Lys-Pro-Val-Ala-His-Val-Val-Ala-Asn-H's-Gln-Val-Glu(SEQ ID NO: 2). This sequence was cloned as a C-terminal fusion into thestreptavidin expression vector pTSA-18F. (Sano, T. and Cantor, C. R.,Biochem. Biophy. Res. Commun., 176:571-577 (1991)). Expression wasperformed in BL21 (DE3)(pLysS) bacteria incubated for 3-6 hours afterinduction with 0.4 mM IPTG. Purification of the protein from inclusionbodies was performed as described by Sano and Cantor. (Sano, T. andCantor, C. R., Proc. Natl. Acad. Sci. USA, 87:142-146 (1990)). Thestreptavidin fusion protein was reacted with biotinylated VLPs at a 3:1weight:weight ratio for 1 hour at room temperature. Particles conjugatedto the streptavidin fusion were purified by centrifugation on a 24%-54%linear sucrose gradient. Preparations of 5 μg VLPs with or withoutattached streptavidin-linked self polypeptide were injected into testand control mice, respectively, three times at two-week intervals. Twoweeks after the final injection, serum samples taken from the animaladministered with the composition that included the self polypeptideshowed evidence for anti-self polypeptide antibodies. In contrast,corresponding antibodies were not detected in serum samples from thecontrol animals.

[0136] It is well established that mouse monoclonal antibodies areuseful as therapeutic agents and as reagents for a variety of basic andapplied studies. However, since most monoclonal antibodies are of mouseor rat origin, the currently available set of antibodies is deficient inthose that specifically recognize mouse or rat epitopes displayed on thesurface of central antigens in their native conformation. Thisdeficiency is limiting for human studies because rodents are models forstudying mammalian biology and highly conserved amino acid sequences ofprotein typically have important functions that are conserved throughevolution. Accordingly, using the methods disclosed herein it will bepossible to stimulate B cell responses against self antigens in theirnative conformation. Thereafter it will be possible to prepare andscreen for hybridomas producing monoclonal antibodies having the desiredbinding specificity. Specifically, the TNF-α VLP that is most effectivein generating polyclonal antibodies against TNF-α will used in anattempt to generate monoclonal antibodies that specifically recognizeand functionally inactivate mouse TNF-α. The standard spleencells/myeloma fusion method will be used to generate the monoclonalantibody producing cells (Galfre et al., Nature, 266:550 (1977)).

[0137] Example 10 briefly describes a method that can be used to producemouse monoclonal antibodies against TNF-α.

EXAMPLE 10 Production of Monoclonal Antibodies

[0138] Mice are first immunized with virus-like particles displaying ontheir surface a mouse TNF-α polypeptide prepared according to theabove-described methods. It is established using an ELISA assay that theserum from the mice contains antibodies specific for native TNF-α. Themice are sacrificed, and harvested spleen cells are fused withnon-secreting myeloma cells to produce a collection of hybridomas. Thehybridomas are screened by methods that will be familiar to those havingordinary skill in the art to identify those secreting antibodies havingbinding specificity for native TNF-α. Useful quantities of theanti-TNF-α antibodies are then purified.

EXAMPLE 11 Increasing Antibody Response to Subdominant VirusNeutralizing Epitopes

[0139] Vaccination with papillomavirus L1 and L1/L2 VLPs can generatehigh titer (greater than 100,000) but only type specific neutralizingantisera (Kirnbauer, R. et al., PNAS, 89:12180-4 (1992); Roden, R. etal., J. Virol., 70:5875-83 (1996)). In contrast, L2 alone or fragmentsthereof elicit only low titers of neutralizing antisera (1000 or less),but they can be cross-neutralizing among papillomavirus types (Roden, R.et al., J. Viro., 68:7570-4 (1994); Kawana, K. et al., J. Virol.,73:6188-90 (1999); Roden, R. et al., Abstract Book, 17^(th)International Papillomavirus Conference, p. 61 (1999)). To increase thetiters of cross-neutralizing L2 antibodies, L2 peptides are displayed ina closely spaced array on the surface of VLPs. For example, a peptideincluding amino acids 108-120 of HPV16 L2 is genetically fused to the Cterminus of streptavidin and the fusion protein is produced as describedin Example 9. The streptavidin-L2 fusion protein is reacted withbiotinylated VLPs and the conjugated VLPs purified as outlined inExample 9. Antisera from streptavidin-L2 conjugated VLP vaccinatedmammals are tested for antibodies that cross-neutralize papillomaviruspseudo virions, using the previously described in vitro neutralizingassays (Roden, R. et al., J. Virol., 68:7570-4 (1994); Roden, R. et al.,Abstract Book, 17_(th) International Papillomavirus Conference, p. 61(1999)). High titers of cross-neutralizing antibodies are detected.

[0140] Although the invention has been described with reference toembodiments and examples, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims. All references cited herein are hereby expressly incorporated byreference.

1 2 1 16 PRT Mus musculus 1 His Tyr Ala Ala Asn Glu Trp Val Phe Gly AsnIle Met Cys Lys Val 1 5 10 15 2 20 PRT Mus musculus 2 Ser Ser Gln AsnSer Ser Asp Lys Pro Val Ala His Val Val Ala Asn 1 5 10 15 His Gln ValGlu 20

What is claimed:
 1. A composition comprising: a support having anordered assembly of subunits; and at least one B cell epitope of atolerogen joined to the support so as to form a tolerogen-presentingimmunogen, wherein the tolerogen-presenting immunogen displays thetolerogen in a regular, repetitive array.
 2. The composition of claim 1,wherein the support is a virus-like particle, a capsomeric structure, ora bead.
 3. The composition of claim 2, wherein the virus-like particle,the capsomeric structure, or the bead comprises a virus capsid protein.4. The composition of claim 3, wherein the virus-like particle, thecapsomeric structure, or the bead comprises a virus capsid protein of anicosohedral virus.
 5. The composition of claim 4, wherein theicosohedral virus is one with T=7 symmetry.
 6. The composition of claim5, wherein the icosohedral virus is a papillomavirus.
 7. A compositioncomprising: a capsomeric structure having a symmetrical assembly ofcapsid proteins; and at least one B cell epitope of a tolerogen joinedto the capsomeric structure so as to form a tolerogen presentingvirus-like particle (VLP), wherein the tolerogen presenting VLP displaysthe tolerogen in an ordered, repetitive array.
 8. The composition ofclaim 7, wherein the capsid protein is a capsid protein from a virusselected from the group consisting of papillomavirinae, polyomavirinae,and parvoviridae.
 9. The composition of claim 7, wherein the capsidprotein is a papillomavirus L1 protein.
 10. The composition of claim 7,wherein the capsomeric structure is icosohedral.
 11. The composition ofclaim 1 or claim 7, wherein the tolerogen is joined to the support by alinker.
 12. The composition of claim 11, wherein the linker comprisesbiotin.
 13. The composition of claim 1 or claim 7, wherein the tolerogenis selected from the group consisting of a peptide, nucleic acid,carbohydrate, and lipid.
 14. The composition of claim 1 or claim 7,wherein the tolerogen is a self antigen.
 15. The composition of claim 1or claim 7, wherein the tolerogen comprises a protein expressed on thesurface of a cell.
 16. The composition of claim 1 or claim 7, whereinthe tolerogen is a protein associated with angiogenesis.
 17. Thecomposition of claim 1 or claim 7, wherein the tolerogen is CCR5. 18.The composition of claim 1 or claim 7, wherein the tolerogen is TumorNecrosis Factor a (TNF-α).
 19. An isolated complex comprising thecomposition of claim 1 or claim 7 joined to a cell of the immune system.20. A pharmaceutical comprising the composition of claim 1 or claim 7.21. A method of generating antibodies to a tolerogen comprising;identifying a subject in need of antibodies to a tolerogen; andproviding to the subject a sufficient amount of the composition of claim1 or claim 7 to generate antibodies to the tolerogen.
 22. The method ofclaim 21, wherein the tolerogen is a self antigen.
 23. A method ofidentifying agents that generate auto-antibodies comprising: (a)providing the composition of claim 1 or claim 7 to a subject; (b)isolating antibodies from the subject; (c) determining the titer of theantibodies isolated in step (b) that bind to the tolerogen; and (d)identifying the agent by the ability to generate high titer antibodies.24. A method of inhibiting HIV infection comprising the step ofadministering the pharmaceutical of claim
 20. 25. A method of reducinginflammation comprising administering the pharmaceutical of claim 20.26. A method of treating chronic viral infection comprisingadministering the pharmaceutical of claim
 20. 27. A method of generatinghigh titer antibodies comprising administering the agent identified bythe method of claim 23 to a subject in need thereof.
 28. A method ofgenerating monoclonal antibodies to a tolerogen comprising: providingthe composition of claim 1 or claim 7 to a subject; and making ahybridoma with a B cell from the subject.
 29. A method of enhancing theproduction of antibodies to a normally immunogenic compound comprising:selecting an antigen that generates a low titer antibody response in asubject; joining this antigen to a modified VLP so as to form aconjugated VLP, wherein the conjugated VLP displays the antigen in aregular repetitive array; and providing the conjugated VLP to a subjectand thereby generating high titer antibodies.
 30. The method of claim 29wherein the spacing between the antigens is above 50 angstroms.
 31. Themethod of claim 29 wherein the modified VLP is joined to the antigen byway of a biotin molecule.